Inflammatory eye disorders

ABSTRACT

Provided herein are methods of evaluating efficacy of a treatment in a subject having eye inflammation (e.g., a subject having dry eye syndrome) and selecting a subject for participation in a clinical study. Also provided are methods of treating a subject having eye inflammation (e.g., a subject having dry eye syndrome).

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.14/379,380, filed on Aug. 18, 2014, which is a U.S. national stage under35 U.S.C. §371 of International Patent Application NumberPCT/US2013/027181, filed on Feb. 21, 2013, which claims priority to U.S.Provisional Patent Application Ser. No. 61/601,149, filed on Feb. 21,2012. The entire contents of the foregoing are incorporated byreference.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under grant number NIHK12-EY016335 and NIH K08-EY020575 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Eye inflammation is common and occurs in humans of all ages. It can lastfrom a few minutes to years, depending on the type and severity of theunderlying disease, disorder, or condition. Non-limiting causes of eyeinflammation include infection (e.g., bacterial, fungal, viral, orparasitic infection), allergy, autoimmune disorders (e.g., ankylosingspondylitis, Behcet's syndrome, dermatomyositis, Graves' disease,juvenile rheumatoid arthritis, multiple sclerosis, psoriatic arthritis,Reiter's syndrome, rheumatoid arthritis, Sjogren's syndrome, systemiclupus erythematosus, and Wegener's granulatomatosis), dry eye syndrome,irritation, and injury or trauma to the eye or eyelid.

Dry eye syndrome is one of the most common eye problems affecting thegeneral population. Dry eye syndrome can cause problems that range inseverity from mildly irritating to debilitating. In healthy subjects,the eye produces a tear film that covers and protects the surface of theeye. The tear film is normally a stable, homogenous layer that providesthe cornea and conjunctiva with protection from the air. Patients withdry eye syndrome lack a sufficient tear film (e.g., lack of tears and/orunstable tears) on some surfaces on the cornea and conjunctiva thatleads to symptoms of irritation and changing vision. Patients having eyeinflammation (e.g., subjects having dry eye syndrome) are commonlyprescribed artificial tears, an oral antibiotic, and/or cyclosporine,and occasionally short-term use of steroids.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that efficacy oftreatment of eye inflammation or dry eye syndrome in a subjectcorrelates with changes in one or more of the following ocular physicalfeatures that can be determined (e.g., using in vivo confocalmicroscopy): (i) a decrease in the number or percentage ofhyperreflective superficial epithelial cells present in the cornea(e.g., in the center of the cornea), (ii) a decrease in the average sizeof superficial epithelial cells present in the cornea (e.g., in thecenter of the cornea), (iii) an elevation in the density of superficialepithelial cells present in the cornea (e.g., in the center of thecornea), (iv) a decrease in the density of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), (v) adecrease in the average size of dendritic immune cells present in thecornea (e.g., in the center of the cornea), (vi) a decrease in theaverage area covered by dendritic immune cells present in the cornea(e.g., in the center of the cornea), (vii) an elevation in the densityor average length of nerves present in the cornea, (viii) an elevationin the amount of branching in nerves present in the cornea, and (ix) anelevation in the total number of nerves present in the cornea (e.g., ascompared to the corresponding levels in the subject prior to treatmentor at an earlier time point in treatment). The invention is also based,in part, on the discovery that subjects having eye inflammation (e.g.,allergy, limbal stem cell insufficiency, or graft versus host disease)have specific ocular physical features (e.g., the number and/or averagedensity of dendritic inflammatory cells present in the peripheralcornea), that can be determined (e.g., using in vivo confocalmicroscopy), and can be used to select a therapy.

In view of these discoveries, provided herein are methods of evaluatingefficacy of a treatment in a subject having eye inflammation (e.g., asubject having dry eye syndrome, a subject having limbal stem celldeficiency, or a subject having graft versus host disease) and selectinga subject for participation in a clinical study. These methods requiredetermining in an eye of a subject or obtaining (or reviewing previouslyobtained or recorded) medical information for the subject regarding oneor more of (i) the number or percentage of hyperreflective superficialepithelial cells present in the cornea (e.g., in the center of thecornea), (ii) the average size of superficial epithelial cells presentin the cornea (e.g., in the center of the cornea), (iii) the density ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), (iv) the density of dendritic immune cells present inthe cornea (e.g., in the center of the cornea), (v) the average size ofdendritic immune cells present in the cornea (e.g., in the center of thecornea), (vi) the average area covered by dendritic immune cells presentin the cornea (e.g., in the center of the cornea), (vii) the density oraverage length of nerves present in the cornea, (viii) the amount ofbranching in nerves present in the cornea, and (ix) the total number ofnerves present in the cornea. In some embodiments, the determining isperformed using in vivo confocal microscopy. In these methods one ormore of the following ocular physical features indicates that thetreatment was effective in the subject: (i) a decrease in the number orpercentage of hyperreflective superficial epithelial cells present inthe center of the cornea, (ii) a decrease in the average size ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), (iii) an elevation in the density of superficialepithelial cells present in the cornea (e.g., in the center of thecornea), (iv) a decrease in the density of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), (v) adecrease in the average size of dendritic immune cells present in thecornea (e.g., in the center of the cornea), (vi) a decrease in theaverage area covered by dendritic immune cells present in the cornea(e.g., in the center of the cornea), (vii) an elevation in the densityor average length of nerves present in the cornea, (viii) an elevationin the amount of branching in nerves present in the cornea, and (ix) anelevation in the total number of nerves present in the cornea (e.g., ascompared to the corresponding levels in the subject prior to treatmentor at an earlier time point in treatment). Some embodiments furtherinclude identifying the treatment administered to a subject having aneye with one or more of the following (i) a decrease in the number orpercentage of hyperreflective superficial epithelial cells present inthe center of the cornea, (ii) a decrease in the average size ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), (iii) an elevation in the density of superficialepithelial cells present in the cornea (e.g., in the center of thecornea), (iv) a decrease in the density of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), (v) adecrease in the average size of dendritic immune cells present in thecornea (e.g., in the center of the cornea), (vi) a decrease in theaverage area covered by dendritic immune cells present in the cornea(e.g., in the center of the cornea), (vii) an elevation in the densityor average length of nerves present in the cornea, (viii) an elevationin the amount of branching in nerves present in the cornea, and (ix) anelevation in the total number of nerves present in the cornea (e.g., ascompared to the corresponding levels in the subject prior to treatmentor at an earlier time point in treatment) as being effective in thesubject.

Also provided are methods of treatment that include selectivelyadministering to an eye(s) of a subject having dry eye syndrome,determined to have an elevated density of dendritic immune cells presentin the center of the cornea as compared to a reference level, a topicalsteroid solution; or selectively topically or orally administering to asubject having dry eye syndrome, determined to have a substantially thesame or a decreased density of dendritic immune cells present in thecenter of the cornea as compared to a reference level, at least twoimmunosuppressive agents. Also provided are methods of using a topicalsteroid solution for treating a subject having dry eye syndromedetermined to have an elevated density of dendritic immune cells presentin the center of the cornea as compared to a reference level. Alsoprovided herein are a topical steroid solution for use in treating asubject having dry eye syndrome determined to have an elevated densityof dendritic immune cells present in the center of the cornea ascompared to a reference level and/or for use in the manufacture of amedicament for treating dry eye syndrome in a subject (e.g., fortreating dry eye syndrome in a subject determined to have an elevateddensity of dendritic immune cells present in the center of the cornea ascompared to a reference level). Also provided are methods of using atleast two immunosuppressive agents (e.g., formulated for topical or oraladministration) for treating a subject having dry eye syndromedetermined to have substantially the same or a decreased density ofdendritic immune cells present in the center of the cornea as comparedto a reference level. Also provided are at least two immunosuppressiveagents (e.g., formulated for topical or oral administration) for use intreating a subject having dry eye syndrome determined to havesubstantially the same or a decreased density of dendritic immune cellspresent in the center of the cornea as compared to a reference leveland/or for use in the manufacture of a medicament for treating dry eyesyndrome in a subject (e.g., for treating dry eye syndrome in a subjectdetermined to have substantially the same or a decreased density ofdendritic immune cells present in the center of the cornea as comparedto a reference level).

Also provided are methods of treating a subject that include selectivelytopically and/or orally administering to a subject, determined to havean elevated number or elevated average density of dendritic inflammatorycells present in the peripheral cornea as compared to a correspondingreference value, at least one anti-inflammatory steroid and/or at leastone immunosuppressive agent (e.g., at least one calcineurin inhibitor).Also provided are methods of using at least one anti-inflammatorysteroid and/or at least one immunosuppressive agent (e.g., at least onecalcineurin inhibitor) (e.g., formulated for topical and/or oraladministration) for treating a subject determined to have an elevatednumber or elevated average density of dendritic inflammatory cellspresent in the peripheral cornea as compared to a correspondingreference value. Also provided are at least one anti-inflammatorysteroid and/or at least one immunosuppressive agent (e.g., at least onecalcinceurin inhibitor) (e.g., formulated for topical and/or oraladministration) for use in treating a subject determined to have anelevated number or elevated average density of dendritic inflammatorycells present in the peripheral cornea as compared to a correspondingreference value and/or for use in the manufacture of a medicament fortreating a subject determined to have an elevated number or elevatedaverage density of dendritic inflammatory cells present in theperipheral cornea as compared to a corresponding reference value.

Also provided are methods of selecting a subject for participation in aclinical study that include determining in an eye of a subject orobtaining (or reviewing previously obtained or recorded) medicalinformation for the subject regarding the number or average density ofdendritic inflammatory cells present in the peripheral cornea. In someembodiments, the determining is performed using in vivo confocalmicroscopy.

Provided herein are methods of evaluating efficacy of a treatment in asubject having eye inflammation (e.g., a subject having dry eyesyndrome, a subject having allergy, a subject having limbal stem celldeficiency, or a subject having graft versus host disease) that include:(a) determining in an eye of a subject having eye inflammation (e.g., asubject having dry eye syndrome, a subject having allergy, a subjecthaving limbal stem cell deficiency, or a subject having graft versushost disease), or alternatively obtaining, providing, or usingpreviously determined information regarding, one or more of (i) thenumber or percentage of hyperreflective superficial epithelial cellspresent in the cornea, (ii) the average size of superficial epithelialcells present in the cornea, (iii) the density of superficial epithelialcells present in the cornea, (iv) the density of dendritic immune cellspresent in the cornea, (v) the average size of dendritic immune cellspresent in the cornea, (vi) the average area covered by dendritic immunecells present in the cornea, (vii) the density or average length ofnerves present in the cornea, (viii) the amount of branching in nervespresent in the cornea, and (ix) the total number of nerves present inthe cornea at a first time point; (b) determining in the eye of thesubject, or alternatively obtaining, providing, or using previouslydetermined information regarding, one or more of (i) the number orpercentage of hyperreflective superficial epithelial cells present inthe cornea, (ii) the average size of superficial epithelial cellspresent in the cornea, (iii) the density of superficial epithelial cellspresent in the cornea, (iv) the density of dendritic immune cellspresent in the cornea, (v) the average size of dendritic immune cellspresent in the cornea, (vi) the average area covered by dendritic immunecells present in the cornea, (vii) the density or average length ofnerves present in the cornea, (viii) the amount of branching in nervespresent in the cornea, and (ix) the total number of nerves present inthe cornea at a second time point; and (c) comparing the one or more of(i) the number or percentage of hyperreflective superficial epithelialcells present in the cornea, (ii) the average size of superficialepithelial cells present in the cornea, (iii) the density of superficialepithelial cells present in the cornea, (iv) the density of dendriticimmune cells present in the cornea, (v) the average size of dendriticimmune cells present in the cornea, (vi) the average area covered bydendritic immune cells present in the cornea, (vii) the density oraverage length of nerves present in the cornea, (viii) the amount ofbranching in nerves present in the cornea, and (ix) the total number ofnerves present in the cornea determined at the first and second timepoints, where (i) the first time point is prior to treatment and thesecond time point is any time point following the initiation oftreatment, or (ii) the first time point is following the initiation oftreatment and the second time point is at a later time point during orafter treatment; and one or more of a decrease in the number orpercentage of hyperreflective superficial epithelial cells present inthe cornea, a decrease in the average size of superficial epithelialcells present in the cornea, an elevation in the density of superficialepithelial cells present in the cornea, a decrease in the density ofdendritic immune cells present in the cornea, a decrease in the averagesize of dendritic immune cells present in the cornea, a decrease in theaverage area covered by dendritic immune cells present in the cornea, anelevation in the density or average length of nerves present in thecornea, an elevation in the amount of branching in nerves present in thecornea, and an elevation in the total number of nerves present in thecornea determined at the second time point compared to the first timepoint indicates that the treatment was effective in the subject, andoptionally, further including (d) identifying the treatment administeredto a subject having in an eye one or more of a decrease in the number orpercentage of hyperreflective superficial epithelial cells present inthe cornea, a decrease in the average size of superficial epithelialcells present in the cornea, an elevation in the density of superficialepithelial cells present in the cornea, a decrease in the density ofdendritic immune cells present in the cornea, a decrease in the averagesize of dendritic immune cells present in the cornea, a decrease in theaverage area covered by dendritic immune cells present in the cornea, anelevation in the density or average length of nerves present in thecornea, an elevation in the amount of branching in nerves present in thecornea, and an elevation in the total number of nerves present in thecornea determined at the second time point compared to the first timepoint, as being effective in the subject. In some embodiments, thesuperficial epithelial cells are present in the center of the cornea. Insome embodiments, the dendritic immune cells are present in the centerof the cornea. In some embodiments, the determining in (a) and (b) isperformed using in vivo confocal microscopy.

Some embodiments further include assessing, or alternatively obtaining,providing, or using previously determined information regarding, one ormore symptoms of dry eye syndrome in the subject at the first and secondtime point. In some embodiments, one or more of (i) the number orpercentage of hyperreflective superficial epithelial cells present inthe cornea, (ii) the average size of superficial epithelial cellspresent in the cornea, and (iii) the density of superficial epithelialcells present in the cornea is determined in the subject or obtained(e.g., obtained from previously recorded medical information) for thesubject, at the first and second time points. In some embodiments, oneor more of (iv) the density of dendritic immune cells present in thecornea, (v) the average size of dendritic immune cells present in thecornea, (vi) the average area covered by dendritic immune cells presentin the cornea is determined in the subject, or alternatively obtaining,providing, or using previously determined information regarding, at thefirst and second time points. In some embodiments, one or more of (vii)the density or average length of nerves present in the cornea, (viii)the amount of branching in nerves present in the cornea, and (ix) thetotal number of nerves present in the cornea is determined in thesubject, or alternatively obtained or provided from or using previouslydetermined information for the subject, at the first and second timepoints.

Some embodiments further include administering to the subject atreatment for eye inflammation (e.g., a treatment for dry eye syndrome).In some embodiments, the subject is a participant in a clinical trial.

Also provided are methods of selecting a subject for participation in aclinical study that include: (a) determining in an eye of a subject, oralternatively obtaining, providing, or using previously determinedinformation regarding, one or more of (i) the number or percentage ofhyperreflective superficial epithelial cells present in the cornea, (ii)the average size of superficial epithelial cells present in the cornea,(iii) the density of superficial epithelial cells present in the cornea,(iv) the density of dendritic immune cells present in the cornea, (v)the average size of dendritic immune cells present in the cornea, (vi)the average area covered by dendritic immune cells present in thecornea, (vii) the density or average length of nerves present in thecornea, (viii) the amount of branching in nerves present in the cornea,and (ix) the total number of nerves present in the cornea; (b) comparingthe one or more of (i) the number or percentage of hyperreflectivesuperficial epithelial cells present in the cornea, (ii) the averagesize of superficial epithelial cells present in the cornea, (iii) thedensity of superficial epithelial cells present in the cornea, (iv) thedensity of dendritic immune cells present in the cornea, (v) the averagesize of dendritic immune cells present in the cornea, (vi) the averagearea covered by dendritic immune cells present in the cornea, (vii) thedensity or average length of nerves present in the cornea, (viii) theamount of branching in nerves present in the cornea, and (ix) the totalnumber of nerves present in the cornea, determined in the eye of thesubject to one or more corresponding reference values; and (c) selectinga subject having one or more of (i) an elevation in the number orpercentage of hyperreflective superficial epithelial cells present inthe cornea, (ii) an elevation in the average size of superficialepithelial cells present in the cornea, (iii) a decrease in the densityof superficial epithelial cells present in the cornea, (iv) an elevationin the density of dendritic immune cells present in the cornea, (v) anelevation in the average size of dendritic immune cells present in thecornea, (vi) an elevation in the average area covered by dendriticimmune cells present in the cornea, (vii) a decrease in the density oraverage length of nerves present in the cornea, (viii) a decrease in theamount of branching in nerves present in the cornea, and (ix) a decreasein the total number of nerves present in the cornea, compared to the oneor more corresponding reference values for participation in a clinicalstudy. In some embodiments, the superficial epithelial cells are presentin the center of the cornea. In some embodiments, the dendritic immunecells are present in the center of the cornea. In some embodiments, thedetermining in (a) is performed using in vivo confocal microscopy.

In some embodiments, the one or more corresponding reference values arethreshold values. In some embodiments, the one or more correspondingreference values are one or more of (i) the number or percentage ofhyperreflective superficial epithelial cells present in the cornea, (ii)the average size of superficial epithelial cells present in the cornea,(iii) the density of superficial epithelial cells present in the cornea,(iv) the density of dendritic immune cells present in the cornea, (v)the average size of dendritic immune cells present in the cornea, (vi)the average area covered by dendritic immune cells present in thecornea, (vii) the density or average length of nerves present in thecornea, (viii) the amount of branching in nerves present in the cornea,and (ix) the total number of nerves present in the cornea, determined in(or recorded or previously observed for) the eye of a healthy subject.In some embodiments, the one or more corresponding reference values aredetermined using in vivo confocal microscopy. In some embodiments, thereference value is determined in a cohort of reference subjects. In someembodiments, the reference value is statistically determined in a cohortof reference subjects, e.g., is the median, mean, or a percentile (e.g.,tertile, quartile, or quintile) cut-off value (e.g., the top percentile,e.g., top tertile, quartile, or quintile cut-off value) in a cohort ofreference subjects.

In some embodiments, the subject is diagnosed as having dry eyesyndrome. In some embodiments, the subject has limbal stem celldeficiency and/or graft versus host disease.

In some embodiments, one or more of (i) the number or percentage ofhyperreflective superficial epithelial cells present in the cornea, (ii)the average size of superficial epithelial cells present in the cornea,(iii) the density of superficial epithelial cells present in the corneais determined in the subject or obtained (e.g., obtained from previouslyrecorded medical information) for the subject. In some embodiments, oneor more of (iv) the density of dendritic immune cells present in thecornea, (v) the average size of dendritic immune cells present in thecornea, (vi) the average area covered by dendritic immune cells presentin the cornea is determined in the subject or obtained (e.g., obtainedfrom previously recorded medical information) for the subject. In someembodiments, one or more of vii) the density or average length of nervespresent in the cornea, (viii) the amount of branching in nerves presentin the cornea, and (ix) the total number of nerves present in the corneais determined in the subject or obtained (e.g., obtained from previouslyrecorded medical information) for the subject.

Also provided are methods of treating a subject having dry eye syndromethat include selectively administering to an eye(s) of a subject havingdry eye syndrome, determined to have an elevated number or density ofdendritic immune cells present in the center of the cornea as comparedto a reference level, a topical steroid solution; or selectively orallyor topically administering to a subject having dry eye syndrome,determined to have no substantial change or a decreased number ordensity of dendritic immune cells present in the center of the cornea ascompared to a reference level, two or more immunosuppressive agents.Also provided are methods of using a topical steroid solution fortreating a subject having dry eye syndrome determined to have anelevated number or density of dendritic immune cells present in thecenter of the cornea as compared to a reference level. Also provided aretopical steroid solutions for use in treating a subject having dry eyesyndrome determined to have an elevated number or density of dendriticimmune cells present in the center of the cornea as compared to areference level and/or for use in the manufacture of a medicament fortreating dry eye syndrome in a subject (e.g., for treating dry eyesyndrome in a subject determined to have an elevated number or densityof dendritic immune cells present in the center of the cornea ascompared to a reference level. Also provided are methods of using two ormore immunosuppressive agents (e.g., formulated for oral or topicaladministration) for treating a subject having dry eye syndromedetermined to have an elevated number or density of dendritic immunecells present in the center of the cornea as compared to a referencelevel. Also provided are two or more immunosuppressive agents (e.g.,formulated for oral or topical administration) for use in treating asubject having dry eye syndrome determined to have an elevated number ordensity of dendritic immune cells present in the center of the cornea ascompared to a reference level and/or for use in the manufacture of amedicament for treating dry eye syndrome in a subject (e.g., fortreating dry eye syndrome in a subject determined to have an elevatednumber or density of dendritic immune cells present in the center of thecornea as compared to a reference level.

In some embodiments, the topical steroid solution contains a steroidselected from the group of: loteprednol etabonate, dexamethasone,hydrocortisone, prednisolone, prednisone, methylprednisolone,betamethasone, dexamethasone, triamcinolone, beclometasone,fludrocortisone, deoxycorticosterone, and aldosterone. In someembodiments, the topical steroid solution contains loteprednoletabonate. In some embodiments, the topical steroid solution isadministered at least once a day.

In some embodiments, at least one of the at least two immunosuppressiveagents is a steroid (e.g., a steroid selected from the group ofloteprednol etabonate, dexamethasone, hydrocortisone, prednisolone,prednisone, methylprednisolone, betamethasone, dexamethasone,triamcinolone, beclometasone, fludrocortisone, deoxycorticosterone, andaldosterone). In some embodiments, at least one of the twoimmunosuppressive agents is selected from the group of pimecrolimus,tacrolimus, sirolimus, and cyclosporine. In some embodiments, at leastone of the at least two immunosuppressive agents is selected from thegroup consisting of pimecrolimus, tacrolimus, sirolimus, andcyclosporine. In some embodiments, the at least two immunosuppressiveagents are administered at least twice a week (e.g., at least once aday).

Some embodiments further include determining, or alternativelyobtaining, providing, or using previously determined informationregarding, the number or average density of dendritic inflammatory cellspresent in the center of the cornea in an eye of the subject, andcomparing the number or average density of dendritic inflammatory cellspresent in the center of the cornea in the eye of the subject to acorresponding reference value. In some embodiments, the determining isperformed using in vitro confocal microscopy. Some embodiments furtherinclude selecting a subject determined to have an elevated number ordensity of dendritic inflammatory cells present in the center of thecornea in the eye of the subject as compared to the correspondingreference value.

Also provided are methods of treating a subject that include selectivelytopically or orally administering to a subject determined to have anelevated number or elevated average density of dendritic inflammatorycells present in the peripheral cornea as compared to a correspondingreference value, at least one anti-inflammatory steroid and/or at leastone immunosuppressive agent. Also provided are methods of using at leastone anti-inflammatory steroid and/or at least one immunosuppressiveagent (e.g., formulated for topical or oral administration) for treatinga subject determined to have an elevated number or elevated averagedensity of dendritic inflammatory cells present in the peripheral corneaas compared to a corresponding reference value. Also provided are atleast one anti-inflammatory steroid and/or at least oneimmunosuppressive agent (e.g., formulated for topical or oraladministration) for use in treating a subject determined to have anelevated number or elevated average density of dendritic inflammatorycells present in the peripheral cornea as compared to a correspondingreference value and/or for use in the manufacture of a medicament fortreating a subject determined to have an elevated number or elevatedaverage density of dendritic inflammatory cells present in theperipheral cornea as compared to a corresponding reference value. Insome embodiments, the subject is diagnosed as having eye inflammation.In some embodiments, the subject has limbal stem cell insufficiency. Insome embodiments, the at least one anti-inflammatory steroid is selectedfrom the group consisting of: hydrocortisone, cortisone, prednisone,prednisolone, methylprednisolone, dexamethasone, betamethasone,triamcinolone, beclometasone, fludrocortisone, deoxycorticosterone, andaldosterone. In some embodiments, the at least one immunosuppressiveagent is a calcineurin inhibitor. In some embodiments, the at least onecalcineurin inhibitor is selected from the group of pimecrolimus,tacrolimus, sirolimus, and cyclosporine.

Some embodiments further include determining, or alternativelyobtaining, providing, or using previously determined informationregarding, the number or average density of dendritic inflammatory cellspresent in the peripheral cornea in an eye of the subject; and comparingthe number or average density of dendritic inflammatory cells present inthe peripheral cornea in the eye of the subject to a correspondingreference value. In some embodiments, the determining is performed usingin vitro confocal microscopy. Some embodiments further include selectinga subject determined to have an elevated number or density of dendriticinflammatory cells present in the peripheral cornea in the eye of thesubject as compared to the corresponding reference value.

Also provided are methods of selecting a subject for participation in aclinical study that include: (a) determining in an eye of a subject, oralternatively obtaining, providing, or using previously determinedinformation regarding, the number or average density of dendriticinflammatory cells present in the peripheral cornea; (b) comparing thenumber or average density of dendritic inflammatory cells present in theperipheral cornea in the eye of the subject to a corresponding referencevalue; and (c) selecting a subject having an elevation in the number oraverage density of dendritic inflammatory cells compared to thecorresponding reference value for participation in a clinical study. Insome embodiments, the corresponding reference value is a thresholdvalue. In some embodiments, the corresponding reference value is thenumber or average density of dendritic inflammatory cells present in theperipheral cornea in an eye of a healthy subject.

In any of the methods described herein, the subject can have eyeinflammation (e.g., a subject having or diagnosed as having dry eyesyndrome, allergy, limbal stem cell deficiency, or graft versus hostdisease). In any of the methods described herein, the subject can be atincreased risk of developing eye inflammation (e.g., a subject atincreased risk of developing dry eye syndrome, limbal stem celldeficiency, graft versus host disease, or allergy, or a subject with ahistory of prior eye inflammation). In any of the methods describedherein, the subject can present with one or more physical symptoms ofeye inflammation that are observable upon examination without the use ofan in vivo confocal microscope (e.g., any of the in vivo confocalmicroscopes described herein or known in the art). In some embodimentsof any of the methods described herein, the subject can be receiving orhave been previously treated or received treatment for eye inflammation(e.g., treatment for dry eye syndrome, limbal stem cell insufficiency,graft versus host disease, or allergy).

By the term “efficacy” or “efficacy of treatment” is meant the abilityof a treatment (e.g., a therapeutic treatment for an eye inflammatorydisorder, e.g., dry eye syndrome) to reduce the number of symptoms of adisease or disorder in a subject (e.g., reduce the number of symptoms ofdry eye syndrome) and/or decrease (e.g., a significant, detectable, orobservable decrease) the severity, frequency, and/or duration of one ormore (e.g., at least two, three, or four) symptoms of a disease ordisorder in a subject (e.g., reduce the severity, frequency, and/orduration of one or more symptoms of dry eye syndrome in a subject).

By the term “dry eye syndrome” is a multifactorial disease of the ocularsurface and tear film characterized by symptoms of discomfort, visualimpairment, and tear film instability. Non-limiting symptoms of dry eyesyndrome include stinging, burning, or scratchy sensation in the eye,stringy mucus in or around the eye, increased eye irritation from windand smoke, eye fatigue after short periods of reading, sensitivity tolight, periods of excessive tearing, blurred vision, red eyes, and painin the eyes. Additional physical symptoms of dry eye syndrome that canbe determined using imaging, e.g., in vivo confocal microscopy aredescribed herein. Additional symptoms of dry eye syndrome are known inthe art (see, e.g., Yao et al., Am. J. Med. 124:1016-1018, 2011). Dryeye syndrome has a variety of different causes. Non-limiting causes ofdry eye syndrome include blepharitis, environmental dryness, menopause,infection, Sjogren's syndrome, graft versus host disease, and trauma(e.g., surgical trauma, e.g., refractive surgery). Dry eye syndrome isalso known as dry eye disease by those in the art.

By the term “hyperreflective” is meant a cell or cellular structure thatreflects more light (e.g., visible light) than a corresponding referencecell or cellular structure (e.g., a cell or cellular structure in an eyeof a subject not having an eye disorder, e.g., a subject not having dryeye syndrome). Non-limiting cellular structures that can behyperreflective include the cytoplasm of a cell or the cell border(e.g., tight junctions). Non-limiting examples of cells that can behyperreflective include superficial epithelial cells (e.g., superficialepithelial cells present in the center of the cornea). Thehyperreflectivity of a cell or cellular structures can be determined invivo, for example, using the in vivo confocal microscopy methodsdescribed herein.

By the term “superficial epithelial cell present in the cornea” is meantan epithelial cell that is present at or near the surface of the cornea.

By the term “center of the cornea” is meant an approximately circulararea having a diameter of less than 5 mm (e.g., a diameter less than 4.5mm, a diameter less than 4 mm, or a diameter of less than 3 mm) from thegeometric center point of the cornea. As used herein, the phrase “in thecornea” may include, e.g., an area corresponding to the center of thecornea.

By the term “peripheral cornea” is meant an area in the cornea thatfalls outside the center of the cornea (as described above).

By the term “in vivo confocal microscopy” is meant the use of a confocalmicroscope to visualize one or more tissue(s) (e.g., cornea), cells(e.g., superficial epithelial cells present in the cornea), and/orcellular substructures (e.g., nerve branching in the cornea) presentwithin a mammal (e.g., a human). Methods of performing in vivo confocalmicroscopy are described herein.

By the term “dendritic immune cell,” “dendritic inflammatory cell,” or“dendritic cell” is meant a bone-marrow derived hyperreflectivecorpuscular cell with tree-like processes. Dendritic immune cells canact as antigen-presenting cells (e.g., they can phagocytose orendocytose an antigen, and transport and present the antigen toT-lymphocyte(s)). The normal (healthy) cornea (e.g., central cornea)contains immature/precursor-type dendritic cells under steady stateconditions; however, these cells can upregulate maturation markers, suchas MHC class II molecules, and can increase in reflectivity and size.

By the term “length of a nerve” or “nerve length” is generally meant thedistance between the cell body (soma) of the nerve cell and the distalend of the axon (end of the axon that is not proximal to the cell body)of the nerve cell, or the distance between (i) a distal end of adendrite (end of a dendrite that is not proximal to the cell body) thatextends from the cell body at a position approximately opposite to theposition in the cell body where the axon extends from the cell body, and(ii) the distal end of the axon of the nerve cell. In some embodiments,the length of a nerve or nerve length can be determined in the cornea ofa subject using in vivo confocal microscopy methods, e.g., methods knownby those skilled in the art or any of the methods described herein. Insome embodiments, nerve length is determined, e.g., by in vivo confocalmicroscopy, and represented as the sum of the length of the nerve fibersobserved per frame, and may be converted into units of microns per mm².

By the term “reference value” is meant a value that is used forcomparative purposes. In some embodiments, a reference value for one ormore of the ocular physical parameters described herein can be athreshold value. In some embodiments, a reference value for the one ormore ocular physical parameters can be a level or value of the one ormore ocular physical parameters measured in a healthy subject (e.g., asubject that does not have dry eye, e.g., does not present with one ormore symptoms of an eye disorder (e.g., dry eye syndrome) or a subjectthat has not been diagnosed as having an eye disorder (e.g., dry eyesyndrome)). Additional examples of reference values are describedherein.

By the term “topical solution” as used in herein is meant apharmaceutically acceptable solution (e.g., buffer) that contains atherapeutically effective amount of one or more (e.g., at least two,three, or four) agents (e.g., one or more topically-active steroids).Non-limiting examples of steroids that can be included in thesesolutions include: loteprednol etabonate, dexamethasone, rimexolone,hydrocortisone, prednisolone, prednisolone acetate, prednisone,methylprednisolone, betamethasone, dexamethasone, triamcinolone,beclometasone, fludrocortisone, deoxycorticosterone, and aldosterone. Insome embodiments, a topical solution can contain a combination of atleast one steroid and at least one calcineurin inhibitor (e.g.,prednisolone and cyclosporine).

By the term “immunosuppressive agent” is meant an agent that inhibits orprevents an activity of the immune system in one or more tissue(s) inthe body of a subject. Non-limiting examples of immunosuppressive agentsinclude antibodies (e.g., fully human or humanized antibodies) thatspecifically bind to CD20, CD25 (e.g., basiliximab or daclizumab), orCD3 (e.g., muromonab); calcineurin inhibitors (e.g., ciclosporin,pimecrolimus, tacrolimus, sirolimus, and/or cyclosporine); interferons(e.g., interferon-β); steroids (e.g., any of the steroids known in theart or described herein); interleukin-1 receptor antagonists;myophenolate mofetil; Prograph®; azathioprine; methotrexate; and/orTNF-α binding proteins (e.g., antibodies and/or soluble TNF-α receptors,e.g., infliximab, etanercept, and/or adalimumab).

By the term “anti-inflammatory steroid” is meant a steroid that reducesinflammation in one or more tissue(s) in a mammal, generally, by bindingto glucocorticoid receptors. Non-limiting examples of anti-inflammatorysteroids include: hydrocortisone, cortisone, prednisone, prednisolone,methylprednisolone, dexamethasone, betamethasone, triamcinolone,beclometasone, fludrocortisone, deoxycorticosterone, and aldosterone. Insome embodiments, an anti-inflammatory steroid can be orallyadministered to a subject.

By the term “calcineurin inhibitor” is meant an agent that mediates asignificant or detectable decrease in the phosphatase activity ofcalcineruin. Non-limiting examples of calcineurin inhibitors includepimecrolimus, tacrolimus, sirolimus, and cyclosporine.

By the term “subject” is meant any mammal (e.g., a human, mice, rat, andrabbit).

Other definitions appear in context throughout this disclosure. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Methods and materials are describedherein for use in the present invention; other, suitable methods andmaterials known in the art can also be used. The materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, sequences, database entries,and other references mentioned herein are incorporated by reference intheir entirety. In case of conflict, the present specification,including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is set of two in vivo confocal microscopic images of superficialepithelial cells in the central cornea of the right eye of a humansubject having dry eye syndrome (human subject #1) prior to treatment.

FIG. 2 is an in vivo confocal microscopic image of superficialepithelial cells in the central cornea of the right eye of human subject#1 at 6-weeks following the initiation of treatment (topical loteprednolfour times a day for four weeks, followed by twice daily administrationof topical loteprednol, daily administration of artificial tears andRefresh PM ointment at bedtime).

FIG. 3 is an in vivo confocal microscopic image of superficialepithelial cells in the central cornea of the left eye of a humansubject having dry eye syndrome (human subject #2) at four weeks afterthe initiation of treatment (autologous serum eight times a day andloteprednol four times a day for four weeks, then loteprednol twice aday for two weeks, then loteprednol once a day for the long-term).

FIG. 4 is an in vivo confocal microscopic image of superficialepithelial cells in the central cornea of the left eye of human subject#2 at 12 weeks after the initiation of therapy (a time point that iseight weeks after the time FIG. 3 was taken).

FIG. 5 is two in vivo confocal microscopic images of superficialepithelial cells in the central cornea of the right eye of a humansubject having dry eye syndrome (human subject #3) at two months afterthe initiation of treatment (artificial tears).

FIG. 6 is two in vivo confocal microscopic images of superficialepithelial cells in the central cornea of the right eye of human subject#3 at 9-months after the initiation of treatment (same treatment asdescribed in FIG. 5; image taken at a time point that is 7-months laterthan the time the image in FIG. 5 was taken).

FIG. 7 is two in vivo confocal microscopic images of dendritic immunecells in the central cornea of right eye of human subject #1 (a subjecthaving dry eye syndrome) prior to treatment.

FIG. 8 is an in vivo confocal microscopic image of dendritic immunecells in the central cornea of the right eye of human subject #1 at6-weeks post-treatment.

FIG. 9 is an in vivo confocal microscopic image of dendritic immunecells in the central cornea of the right eye of human subject #2 (asubject having dry eye syndrome) at an early time point in treatment.

FIG. 10 is an in vivo confocal microscopic image of dendritic immunecells in the central cornea of the right eye of human subject #2 (asubject having dry eye syndrome) at 8-weeks post-treatment (a time pointthat is later than the time the image in FIG. 9 was taken).

FIG. 11 is two in vivo confocal microscopic images of corneal subbasalnerves in the right eye of human subject #1 (a subject having dry eyesyndrome) prior to treatment. The depth of the image on the left is 51μm, and the depth of the image on the right is 55 μm.

FIG. 12 is an in vivo confocal microscopic image of corneal subbasalnerves in the right eye of human subject #1 at 6-weeks post-treatment.The depth of the image is 65 μm.

FIG. 13 is an in vivo confocal microscopic image of corneal subbasalnerves in the left eye of human subject #2 (a subject having dry eyesyndrome) at an early time point in treatment. The depth of the image is53 μm.

FIG. 14 is two in vivo confocal microscopic images of corneal subbasalnerves in the left eye of human subject #2 at 8-weeks post-treatment (atime point that is later than the time the image in FIG. 13 was taken).The depth of the image on the left is 67 μm, and the depth of the imageon the right is 81 μm.

FIG. 15 is a flow chart showing the therapeutic treatments providedherein that include the selective administration of one or moretherapeutic agents to a subject having dry eye syndrome on the basis ofthe number of hyperreflective superficial epithelial cells present inthe central cornea, the density of dendritic immune cells in the centralcornea, and the density of nerves in the cornea (as determined using invivo confocal microscopy) as compared to a corresponding referencevalue. The therapeutic treatments shown can be cumulative (e.g., asubject having an elevated or “high” number of hyperreflectivesuperficial epithelial cells and an elevated or “high” density ofdendritic immune cells in the central cornea (as compared tocorresponding reference values) can be administered artificial tears incombination with a topical steroid solution).

FIG. 16 is three in vivo confocal microscopic images of superficialepithelial cells in the central cornea of a normal subject (left image),a subject having acute allergy (center image), and a subject havingchronic allergy (right image). These data show that patients withallergy have an elevated level of epithelial cell border reflectivity(change in tight junctions), an elevated level of epithelial cellreflectivity (squamous metaplasia), and an elevation in the averageepithelial cell size in the central cornea as compared to a normal(healthy) subject.

FIG. 17 is three in vivo confocal microscopic images of epithelial cellsin the conjunctiva of a normal subject (left image), a subject havingacute allergy (center image), and a subject having chronic allergy(right image). The depth of the right image is 39 μm. These data showthat patients with allergy have an elevated level of epithelial cellborder reflectivity (change in tight junctions) in the conjunctiva ascompared to a normal (healthy) subject.

FIG. 18 is three in vivo confocal microscopic images of dendritic cellsin the conjunctiva of a normal subject (left image), a subject havingacute allergy (center image), and a subject having chronic allergy(right image). The depth of the right image is 53 μm. These data showthat patients with allergy have an elevated dendritic cell density, anelevation in the average dendritic cell size, and an elevation in thelevel of dendritic cell reflectivity in the conjunctiva as compared to anormal (healthy) subject.

FIG. 19 is two in vivo confocal microscopic images of blood vessels inthe conjunctiva of a normal subject (left image) and a subject havingacute allergy (right image). The data show that a subject having acuteallergy has an elevation in the diameter of the lumen of blood vessels,an elevation in the average inflammatory cell size, and an elevation inthe sticking of inflammatory cells to the blood vessel wall (nomovement) in the conjunctiva as compared to a normal (healthy) subject.

FIG. 20 is two in vivo confocal microscopic images of lymph vessels inthe conjunctiva of a normal subject (left image) and a subject havingacute allergy (right image). The data show that a subject having acuteallergy has an elevation in the number of inflammatory cells present inthe lymph vessel wall and an elevation in the average size ofinflammatory cells in the lymph vessels of the conjunctiva as comparedto a normal (healthy) subject.

FIG. 21 is two in vivo confocal microscopic images of dendritic cells inthe cornea of a normal subject (left image) and a subject having acuteallergy (right image). The data show that a subject having an acuteallergy has an elevation in the density of dendritic cells, an elevationin the average dendritic cell size, and an elevation in the level ofdendritic cell reflectivity in the cornea as compared to a normal(healthy) subject.

FIG. 22 is two in vivo confocal microscopic images of dendritic cells inthe central cornea of a normal subject (left image) and a subject havingacute allergy (right image). The data show that a subject having acuteallergy has an elevation in the average dendritic cell size in thecentral cornea as compared to a healthy subject.

FIG. 23 is a set of six confocal microscopic images showing the cornealepithelium and the subbasal nerve plexus in patients with dry eyesyndrome. The top left image shows superficial epithelial cells in thenormal eye. The top center image shows superficial epithelial cells in asubject having dry eye syndrome. The top right image shows epithelialcell counting in a subject having dry eye syndrome. The bottom leftimage shows the sub-basal nerve plexus in a normal subject. The bottomcenter image is the sub-basal nerve plexus in a subject having dry eyesyndrome. The bottom right image shows nerve density counting in asubject having dry eye syndrome using Image J software.

FIG. 24 is a graph showing the total nerve length in control subjectsand subjects having dry eye syndrome. The error bars represent thestandard deviation from the mean.

FIG. 25 is a graph showing the total number of corneal nerves in controlsubjects and subjects having dry eye syndrome. The error bars representthe standard deviation from the mean.

FIG. 26 is a graph showing the number of corneal main nerve trunks incontrol subjects and subjects having dry eye syndrome. The error barsrepresent the standard deviation from the mean.

FIG. 27 is a graph showing the number of corneal nerve branches incontrol subjects and subjects having dry eye syndrome. The error barsrepresent the standard deviation from the mean.

FIG. 28 is a graph showing the corneal superficial epithelial celldensity in control subjects and subjects having dry eye syndrome. Theerror bars represent the standard deviation of the mean.

FIG. 29 is a graph showing the corneal superficial epithelial cell sizein control subjects and subjects having dry eye syndrome. The error barsrepresent the standard deviation of the mean.

FIG. 30 is a graph showing the corneal basal epithelial density incontrol subjects and subjects having dry eye syndrome. The error barsrepresent the standard deviation of the mean.

FIG. 31 is a linear regression analysis of the relationship betweencorneal total nerve length and corneal superficial epithelial cell area.

FIG. 32 is a linear regression analysis of the relationship between thetotal number of corneal nerves and the corneal superficial epithelialarea.

FIG. 33 is a linear regression analysis of the relationship between thenumber of corneal main nerves and the corneal superficial epithelialcell area.

FIG. 34 is a linear regression analysis of the relationship between thenumber of corneal nerve branches and the corneal superficial epithelialcell area.

FIG. 35 is a linear regression analysis of the relationship between thenumber of corneal nerve branches and the corneal superficial celldensity.

FIG. 36 is a linear regression analysis of the relationship between thetotal corneal nerve length and the corneal basal epithelial celldensity.

FIG. 37 is a linear regression analysis of the relationship between thecorneal subbasal epithelial cell area and fluorescein staining score.

FIG. 38 is a linear regression analysis of the relationship between thecorneal basal epithelial cell density and fluorescein staining score.

FIG. 39 is a linear regression analysis of the relationship between thecorneal superficial epithelial cell density and fluorescein stainingscore.

FIG. 40 is a set of three graphs showing the mean TBUT, mean cornealfluorescein staining score, or the mean Ocular Surface Disease Index(OSDI) score determined for subjects having dry eye syndrome at thefollowing time points: before anti-inflammatory treatment (Pre-Rx), at afirst follow-up visit following initiation of anti-inflammatorytreatment (2.3±0.9 months after initiation of treatment), and at asecond follow-up visit following initiation of anti-inflammatorytreatment (4.8±1.8 months following initiation of anti-inflammatorytreatment).

FIG. 41 is set of three linear regression analyses, from left to right,of the relationship between corneal epithelium immune cell density(cells/mm²) and TBUT, the relationship between intraglandular immunecells (% lumen occupied) and TBUT, and the relationship between acinardensity (acini/mm²) and corneal fluorescein staining score (Oxfordscale) in subjects having dry eye syndrome.

FIG. 42 is a set of four in vivo confocal microscopy corneal images from(A) an eye from a healthy subject, (B) an eye from a subject havingevaporate dry eye syndrome, (C) an eye from a subject having mixedmechanism dry eye syndrome, and (D) an eye from a subject havingaqueous-deficient dry eye syndrome.

FIG. 43 is a graph of the mean dendritic cell corneal density(cells/mm²) for eyes from normal subjects (Normal), eyes from subjectshaving evaporative dry eye syndrome (Evaporative), eyes from subjectshaving mixed mechanism dry eye syndrome (Mixed), and eyes from subjectshaving aqueous-deficient dry eye syndrome (Aqueous-deficient). *,p<0.05; **, p<0.01.

FIG. 44 is a graph of the mean dendritic cell area (μm) in the corneasof eyes from normal subjects (Normal), eyes from subjects havingevaporative dry eye syndrome (Evaporative), eyes from subjects havingmixed mechanism dry eye syndrome (Mixed), and eyes from subjects havingaqueous-deficient dry eye syndrome (Aqueous-deficient). *, p<0.05; **,p<0.01.

FIG. 45 is a graph of the mean area of the cornea covered by dendriticcells (μm²) for eyes from normal subjects (Normal), eyes from subjectshaving evaporative dry eye syndrome (Evaporative), eyes from subjectshaving mixed mechanism dry eye syndrome (Mixed), and eyes from subjectshaving aqueous-deficient dry eye syndrome (Aqueous-deficient). *,p<0.05; **, p<0.01.

FIG. 46 is a graph of the mean number of dendritic processes per cell inthe corneas of eyes from normal subjects (Normal), eyes from subjectshaving evaporative dry eye syndrome (Evaporative), eyes from subjectshaving mixed mechanism dry eye syndrome (Mixed), and eyes from subjectshaving aqueous-deficient dry eye syndrome (Aqueous-deficient). *,p<0.05; **, p<0.01.

FIG. 47 is a set of four graphs showing the mean immune cell density(cells/mm²), mean immune cell area (μm²), mean immune cell area covered(μm²), and mean number of dendrites (dendrites/cell) of immune cellspresent in the cornea of subjects having dry eye syndrome that areeither receiving anti-inflammatory treatment (Anti-inflammatory) or notreceiving an anti-inflammatory treatment (Non Anti-inflammatory).

FIG. 48 is a graph of the mean total nerve length (μm/frame) detected inthe cornea of normal control patients and patients having dry eyesyndrome at baseline (prior to treatment with autologous serum) (*,p<0.001).

FIG. 49 is a graph of the mean percent change in corneal nerves overtime in patients having dry eye syndrome following the initiation oftreatment with autologous serum.

FIG. 50 is a set of two in vivo confocal microscopic images from twodifferent subjects having dry eye syndrome at baseline (prior totreatment with autologous serum).

FIG. 51 is a set of two in vivo confocal microscopic images: one showingthe cornea of a subject having dry eye syndrome at baseline (prior totreatment with autologous serum) (left), and one showing the cornea ofthe same subject following 6 months of treatment with autologous serum(right).

FIG. 52 is a graph showing the mean total nerve length (μm/frame) in thecorneas of normal control subjects (Normal controls), corneas ofpatients having dry eye syndrome at baseline (prior to treatment withautologous serum) (DES Baseline), and corneas of patients having dry eyesyndrome after treatment with autologous serum (DES Post-serum) (

, p<0.001; *, p<0.001).

FIG. 53 is a graph showing the mean total nerve number in the corneas ofnormal control subjects (Normal controls), corneas of patients havingdry eye syndrome at baseline (prior to treatment with autologous serum)(DES Baseline), and corneas of patients having dry eye syndrome aftertreatment with autologous serum (DES Post-serum) (

, p<0.001; *, p<0.001).

FIG. 54 is a graph showing the mean main nerve trunk length (μm/frame)in the corneas of normal control subjects (Normal controls), corneas ofpatients having dry eye syndrome at baseline (prior to treatment withautologous serum) (DES Baseline), and corneas of patients having dry eyesyndrome after treatment with autologous serum (DES Post-serum) (

, p<0.001; *, p<0.001).

FIG. 55 is a graph showing the mean main nerve trunk number in thecorneas of normal control subjects (Normal controls), corneas ofpatients having dry eye syndrome at baseline (prior to treatment withautologous serum) (DES Baseline), and corneas of patients having dry eyesyndrome after treatment with autologous serum (DES Post-serum) (

, p<0.001; *, p<0.001).

FIG. 56 is a graph of the mean nerve branch length in the corneas ofnormal control subjects (Normal controls), corneas of patients havingdry eye syndrome at baseline (prior to treatment with autologous serum)(DES Baseline), and corneas of patients having dry eye syndrome aftertreatment with autologous serum (DES Post-serum) (

, p<0.001; *, p<0.001).

FIG. 57 is a graph of the mean nerve branch number in the corneas ofnormal control subjects (Normal controls), corneas of patients havingdry eye syndrome at baseline (prior to treatment with autologous serum)(DES Baseline), and corneas of patients having dry eye syndrome aftertreatment with autologous serum (DES Post-serum) (

, p<0.001; *, p<0.001).

FIG. 58 is a linear regression comparing the total nerve length inpatients having dry eye syndrome after treatment with autologous serum(Post-serum) to the total nerve length in the same set of patients atbaseline (prior to treatment with autologous serum; Pre-Serum).

FIG. 59 is a graph showing the percentage of patients having dry eyesyndrome that were treated with autologous serum that achieved aspecific percentage of nerve increase following treatment withautologous serum (as compared to the baseline value for each patient).

FIG. 60 is a linear regression comparing the percent nerve changeachieved in each patient having dry eye syndrome following treatmentwith autologous serum compared to the age of each subject.

FIG. 61 is a graph of the mean percent nerve change observed followingtreatment with autologous serum in subjects having evaporative dry eyesyndrome (Evaporative), aqueous dry eye syndrome (Aqueous), mixed dryeye syndrome (Mixed), graft-versus-host disease dry eye syndrome (GCHD),and neurotrophic keratopathy dry eye syndrome (NTK).

FIG. 62 is a graph of the mean TBUT score of patients having dry eyesyndrome at baseline (prior to treatment with autologous serum) andfollowing treatment with autologous serum (Post-serum).

FIG. 63 is a graph of the percentage of patients having dry eye syndromethat received treatment with autologous serum that show a specificpercent increase in TBUT.

FIG. 64 is a graph of the mean OSDI Questionnaire Score for subjectshaving dry eye syndrome at baseline (before treatment with autologousserum; Baseline) and after treatment with autologous serum (Post-serum).

FIG. 65 is a graph of the percentage of patients having dry eye syndromethat were treated with autologous serum that show a specific percentagedecrease in OSDI Questionnaire Score (as compared to the OSDIQuestionnaire Score of each subject prior to treatment with autologousserum).

FIG. 66 is a graph showing the mean percentage of superficial epithelialcells that are reflective in the cornea and the conjunctiva of controlsubjects, subjects having ocular allergy (OA), and subjects havingnon-allergic ocular inflammatory diseases (OID) (*, p<0.0001).

FIG. 67 is a graph showing the mean percentage of superficial epithelialcells that show border reflectivity in the cornea and conjunctiva ofcontrol subjects, subjects having OA, and subjects having OID (*,p<0.0001).

FIG. 68 is a graph showing the mean dendritic cell density in the corneaand conjunctiva of control subjects, subjects having OA, and subjectshaving OID (*, p>0.05).

FIG. 69 is a graph showing the mean dendritic cell density in theconjunctiva of control subjects, subjects having OA, and subjects havingOID (*, p<0.001).

FIG. 70 is a set of twelve exemplary in vivo confocal microscopy imagesshowing the corneal epithelium, conjunctival epithelium, central cornealdendritic cells, and conjunctival dendritical cells in control subjects(Normal), subjects having OA (Allergic), and subjects having OID(Non-Allergic Inflammatory).

FIG. 71 is a set of three exemplary in vivo confocal microscopic imagesshowing the conjunctival blood vessels in a control subject, a subjecthaving OA, and a subject having an OID.

FIG. 72 is graph of the mean dendritic cell density in the centralcornea of each eye in three limbal stem cell insufficiency patientsbefore treatment and after treatment, and in the central corneas ofcontrol patients.

FIG. 73 is an in vivo confocal microscopic image showing the centralcornea of a subject having limbal stem cell insufficiency beforetreatment (pre-treatment) (left) and an in vivo confocal microscopicimage showing the central cornea of the same subject after 3-months oftreatment (3 months on treatment) (right).

FIG. 74 is a graph of the mean nerve density in the central cornea ofeach eye in three limbal stem cell insufficiency patients beforetreatment and after treatment, and in the corneas of control patients.

FIG. 75 is a graph of the of the mean nerve count in the central corneaof each eye in three limbal stem cell insufficiency patients beforetreatment and after treatment, and in the corneas of control patients.

FIG. 76 is an in vivo confocal microscopic image of the cornea of asubject having limbal stem cell insufficiency before treatment (left)and an image of the cornea of the same subject after treatment (right).

FIG. 77 is a set of two in vivo confocal microscopic images showing theconjunctivalization in the cornea of a subject having limbal stem cellinsufficiency at presentation (left) and after 4-months of treatment(right).

FIG. 78 is an in vivo confocal microscopic image of the limbal area of ahealthy (control) subject (left) and an in vivo confocal microscopicimage of the peripheral cornea of a healthy subject (right).

FIG. 79 is set of exemplary in vivo confocal microscopic images showingthe central cornea and the peripheral quadrants of the cornea (superior,temporal, inferior, and nasal quandrants) in a healthy (control)subject.

FIG. 80 is a graph showing the mean dendritic cell density in the centercornea, inferior quadrant of the cornea, the superior quadrant of thecornea, the nasal quadrant of the cornea, the temporal quadrant of thecornea, the peripheral areas of the cornea, and the total areas of thecornea of healthy (control) subjects. *, ANOVA with Bonferronicorrection (p<0.0001) for peripheral areas versus central cornea.

FIG. 81 is a graph showing the mean immune cell density by area in thecenter cornea, inferior quadrant of the cornea, the superior quadrant ofthe cornea, the nasal quadrant of the cornea, the temporal quadrant ofthe cornea, the peripheral areas of the cornea, and the total areas ofthe cornea of healthy (control) subjects. No statistical difference wasdetected when comparing all areas.

FIG. 82 is a graph of the mean main nerve trunk number (number/frame) inthe central cornea and in each of the four peripheral cornea quadrants(inferior, nasal, superior, and temporal quadrants) in healthy (control)subjects. The data shown are the mean±SEM.

FIG. 83 is a graph of the mean main nerve trunk length (μm/mm²) in thecentral cornea and in each of the four peripheral cornea quadrants(inferior, nasal, superior, and temporal quadrants) in healthy (control)subjects. The data shown are the mean±SEM.

FIG. 84 is a graph of the mean nerve branch number (number/frame) in thecentral cornea and in each of the four peripheral cornea quadrants(inferior, nasal, superior, and temporal quadrants) in healthy (control)subjects. The data shown are the mean±SEM.

FIG. 85 is a graph of the mean nerve branch length (μm/mm²) in thecentral cornea and in each of the four peripheral cornea quadrants(inferior, nasal, superior, and temporal quadrants) in healthy (control)subjects. The data shown are the mean±SEM.

FIG. 86 is a graph of the mean total nerve number (number/frame) in thecentral cornea and in each of the four peripheral cornea quadrants(inferior, nasal, superior, and temporal quadrants) in healthy (control)subjects. The data shown are the mean±SEM.

FIG. 87 is a graph of the mean total nerve length (μm/mm²) in thecentral cornea and in each of the four peripheral cornea quadrants(inferior, nasal, superior, and temporal quadrants) in healthy (control)subjects. The data shown are the mean±SEM.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, at least in part, on the discovery that efficacyof treatment of eye inflammation (e.g., treatment of dry eye syndrome,graft versus host disease, limbal stem cell insufficiency, or allergy)in a subject correlates with changes in one or more of the followingocular physical features that can be determined (e.g., using in vivoconfocal microscopy): (i) the number or percentage of hyperreflectivesuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), (ii) the average size of superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea), (iii) thedensity of superficial epithelial cells present in the cornea (e.g., inthe center of the cornea), (iv) the density of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), (v) theaverage size of dendritic immune cells present in the cornea (e.g., inthe center of the cornea), (vi) the average area covered by dendriticimmune cells present in the cornea (e.g., in the center of the cornea),(vii) the density or average length of nerves present in the cornea,(viii) the amount of branching in nerves present in the cornea, and (ix)the total number of nerves present in the cornea. The invention is alsobased, in part, on the discovery that subjects having eye inflammation(e.g., subjects having allergy, limbal stem cell insufficiency, graftversus host disease, or dry eye syndrome) have specific physicalfeatures (e.g., the number and/or average density of dendriticinflammatory cells present in the peripheral cornea), that can bedetermined (e.g., using in vivo confocal microscopy), and can be used toselect a therapy. In some embodiments, the subjects has not beenidentified as having any one particular ocular disease. In someembodiments, the subject can have one or more different ocularconditions (e.g., one or more of dry eye syndrome, allergy, infection,limbal stem cell insufficiency, or graft versus host disease).

In view of these discoveries, provided herein are methods of evaluatingefficacy of a treatment in a subject having eye inflammation (e.g., asubject having dry eye syndrome, limbal stem cell insufficiency,allergy, or graft versus host disease) and selecting a subject forparticipation in a clinical study. These methods require determining inan eye of a subject one or more of (i) the number or percentage ofhyperreflective superficial epithelial cells present in the cornea(e.g., in the center of the cornea), (ii) the average size ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), (iii) the density of superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea), (iv) thedensity of dendritic immune cells present in the cornea (e.g., in thecenter of the cornea), (v) the average size of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), (vi) theaverage area covered by dendritic immune cells present in the cornea(e.g., in the center of the cornea), (vii) the density or average lengthof nerves present in the cornea, (viii) the amount of branching innerves present in the cornea, and (ix) the total number of nervespresent in the cornea. Also provided are methods of selecting a subjectfor participation in a clinical study that include determining in an eyeof a subject the number or average density of dendritic inflammatorycells present in the peripheral cornea. In some embodiments of themethods described herein, the determining is performed using in vivoconfocal microscopy.

Also provided are methods of treatment that include selectivelyadministering to an eye(s) of a subject having eye inflammation (e.g., asubject having dry eye syndrome), determined to have an elevated densityof dendritic immune cells present in the center of the cornea ascompared to a reference level, a topical steroid solution; orselectively orally or topically administering to a subject having eyeinflammation (e.g., a subject having dry eye syndrome), determined tohave no substantial change or a decreased density of dendritic immunecells present in the center of the cornea as compared to a referencelevel, at least two immunosuppressive agents. Also provided are methodsof treating a subject that include selectively orally or topicallyadministering to a subject, determined to have an elevated number orelevated average density of dendritic inflammatory cells present in theperipheral cornea as compared to a corresponding reference value, atleast one anti-inflammatory steroid and/or at least oneimmunosuppressive agent (e.g., at least one calcineurin inhibitor).

Eye Inflammation

Eye inflammation is common and occurs in humans of all ages. It can lastfrom a few minutes to years, depending on the type and severity of theunderlying disease, disorder, or condition. Inflammation can occur inone or both eyes at a time, and it may be accompanied by symptomsincluding itching, excessive tear production, and/or eye discharge.Additional non-limiting symptoms of eye inflammation include pain,redness, swelling, tearing, and unusual warmth or heat. Inflammation canbe caused by a variety of different causes. Non-limiting causes of eyeinflammation include infection (e.g., bacterial, fungal, viral, orparasitic infection) (e.g., blepharitis, chalazion, conjunctivitis,dacryocystitis, iritis, keratitis, periorbital cellulitis, scleritis,sinusitis, and stye or bordeolum), allergy (e.g., drug allergies, foodallergies, hay fever or allergic reaction to an allergen, and insectbite allergy)(e.g., chronic or acute allergy), autoimmune disorders(e.g., ankylosing spondylitis, Behcet's syndrome, dermatomyositis,Graves' disease, juvenile rheumatoid arthritis, multiple sclerosis,psoriatic arthritis, Reiter's syndrome, rheumatoid arthritis, Sjogren'ssyndrome, systemic lupus erythematosus, and Wegener's granulatomatosis),graft versus host disease, dry eye syndrome, limbal stem cellinsufficiency, irritation, and injury or trauma to the eye or eyelid(e.g., blunt trauma, corneal abrasion or ulcer, foreign objects ormaterials, hematoma, insect bite or sting, irritants, and orbital bonefracture). The treatment of eye inflammation typically administered to asubject will depend on the underlying cause of the disease. In someembodiments, the treatment can be, e.g., one or more of an eye lubricant(e.g., liquid or ointment), oral or topical antibiotic, an allergytreatment (e.g., an anti-histamine or cromolyn), and/or animmunosuppressive agent (e.g., a topical steroid or cyclosporine). Thetreatment prescribed to a patient, or the efficacy of a prescribedtreatment, can depend upon the identification of the cause of the eyeinflammation in the subject.

Subjects can be diagnosed as having eye inflammation by a medicalprofessional (e.g., a physician, a physician's assistant, a nurse, anurse's assistant, or a laboratory technician). In any of the methodsdescribed herein, the subject can be a child, a teenager, or an adult(e.g., at least 18, 25, 30, 40, 50, 60, 70, 80, or 90 years old). Thesubject can be a male or a female. A subject diagnosed as having eyeinflammation may present with one or more (e.g., two, three, four, five,six, seven, eight, nine, and ten) of the symptoms of eye inflammationdescribed herein. In some embodiments, a subject having eye inflammation(e.g., a low level of inflammation) may not present with a symptom ofeye inflammation that can be easily detected by basic examination of aneye(s) of the subject (examination of the patient that does not involvethe magnification of the tissues of the eye). In some embodiments, thesubject can be diagnosed as having eye inflammation based, in part, onthe detection of one or more (e.g., two, three, four, five, six, seven,eight, nine, or ten) of the ocular physical parameters described herein.For example, the subject can be diagnosed as having eye inflammationbased on the detection of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following ocular physicalparameters (e.g., using in vivo confocal microscopy): an elevation inthe number or percentage of hyperreflective superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea), an elevationin the average size of superficial epithelial cells present in thecornea (e.g., in the center of the cornea), a decrease in the density ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), an elevation in the density of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), an elevationin the average size of dendritic immune cells present in the cornea(e.g., in the center of the cornea), an elevation in the average areacovered by dendritic immune cells present in the cornea (e.g., in thecenter of the cornea), a decrease in the density or average length ofnerves present in the cornea, a decrease in the amount of branching innerves present in the cornea, a decrease in the total number of nervespresent in the cornea, an elevation in the number or average density ofdendritic inflammatory cells present in the peripheral cornea, anelevation in the hyperreflectivity of epithelial cells in theconjunctiva, an elevation in dendritic cell density in the conjunctiva,an elevation in the average dendritic cell size in the conjunctiva, anelevation in the number of hyperreflective dendritic cells in theconjunctiva, an elevation in the dilation of the lumen of blood vesselsin the conjunctiva, an elevation in the average size of inflammatorycells in the blood vessels in the conjunctiva, an elevation in thesticking (elevation in the average time of transitory residence) ofinflammatory cells to the blood vessels walls in the conjunctiva, anelevation in the average size of inflammatory cells in the lymph vesselsin the conjunctiva, an elevation in the number of inflammatory cellspresent in the lymph vessels of the conjunctiva, as compared to areference level of the one or more ocular physical parameters. In someembodiments, the subject may be identified as being at increased risk ofdeveloping eye inflammation (e.g., at increased risk of developing dryeye disorder, allergy, graft versus host disease, or limbal stem celldeficiency). In some embodiments, the subject may be suspected of havingeye inflammation (e.g., eye inflammation caused by dry eye disorder,allergy, limbal stem cell deficiency, graft versus host disease, or anyof the other causes of eye inflammation described herein). In someembodiments, the subject may have a history of previous eyeinflammation. In some embodiments, the subject may be receiving atreatment or have previously received a treatment for eye inflammation.

The reference level as described herein can be a threshold level or canbe a level of the one or more ocular physical parameters in a healthysubject (e.g., a subject that does not have one or more symptoms of eyeinflammation, or a subject that has not been diagnosed as having eyeinflammation or an eye disorder) or the same subject at an earlier timepoints. Non-limiting exemplary threshold values for the density ofhyperreflective superficial epithelial cells present in the cornea(e.g., in the center of the cornea) are 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165cells/mm², or ranges of 65-75, 75-85, 85-95, 95-105, 105-115, 115-125,125-135, 135-145, 145-155, or 155-165 cells/mm². Non-limiting examplesof threshold values for the average size of superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea) are 360, 365,370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435,440, 445, 450, 455, or 460 μm², or ranges of 360-370, 370-380, 380-390,390-400, 400-410, 410-420, 420-430, 430-440, 440-450, or 450-460 μm².Non-limiting examples of threshold values for the density of superficialepithelial cells present in the cornea (e.g., in the center of thecornea) are 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250,2300, 2350, 2400, 2450, or 2500 cells/mm², or ranges of 1800-1900,1900-2000, 2000-2100, 2100-2200, 2200-2300, 2300-2400, or 2400-2500cells/mm². Non-limiting examples of threshold values for the length ofnerves present in the cornea are a total nerve length of 9000, 10000,11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000,21000, or 22000 μm/mm², or ranges of 9000-10000, 10000-11000,11000-12000, 12000-13000, 13000-14000, 14000-15000, 15000-16000,17000-18000, 18000-19000, 19000-20000, 20000-21000, or 21000-22000μm/mm². Non-limiting examples of threshold values for the amount ofbranching in nerves present in the cornea are 4, 5, 6, 7, 8, 9, 10, or11 total number of nerve branches per frame (460 μm×345 μm frame), orranges of 4-11, 4-10, 5-10, 5-8, or 6-11 total number of nerve branchesper frame (460 μm×345 μm frame). Non-limiting examples of thresholdvalues for the total number of nerves present in the cornea are 9, 10,11, 12, 13, 14, 15, or 16 total number of nerves present per frame (460μm×345 μm frame), or ranges of 9-16, 10-16, 10-15, 11-15, 9-12, or 12-16total number of nerves per frame (460 μm×345 μm frame). Non-limitingthreshold values for the density of dendritic immune cells in the cornea(e.g., in the center of the cornea) are 0, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, or 70 cells/mm², or the ranges of 0-10, 10-20,20-30, 30-40, 40-50, 50-60, 60-70, 10-50, or 20-70 cells/mm². Additionalthreshold values can be determined using methods known in the art orthose described herein.

The methods for determining the above ocular physical parameters aredescribed herein. A physician can also monitor a subject for thedevelopment of eye inflammation by assessing one or more of theseparameters at different points over time (e.g., at least once every sixmonths, at least once a year, at least once every two years, and atleast once every three years).

A subject determined to have one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen) of: an elevation in thenumber or percentage of hyperreflective superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea), an elevationin the average size of superficial epithelial cells present in thecornea (e.g., in the center of the cornea), a decrease in the density ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), an elevation in the density of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), an elevationin the average size of dendritic immune cells present in the cornea(e.g., in the center of the cornea), an elevation in the average areacovered by dendritic immune cells present in the cornea (e.g., in thecenter of the cornea), a decrease in the density or average length ofnerves present in the cornea, a decrease in the amount of branching innerves present in the cornea, a decrease in the total number of nervespresent in the cornea, an elevation in the number or average density ofdendritic inflammatory cells present in the peripheral cornea, anelevation in the reflectivity of epithelial cells in the conjunctiva, anelevation in dendritic cell density in the conjunctiva, an elevation inthe average dendritic cell size in the conjunctiva, an elevation in thenumber of hyperreflective dendritic cells in the conjunctiva, anelevation in the dilation of the lumen of blood vessels in theconjunctiva, an elevation in the average size of inflammatory cells inthe blood vessels in the conjunctiva, an elevation in the sticking(elevation in the average time of transitory residence) of inflammatorycells to the blood vessels walls in the conjunctiva, an elevation in theaverage size of inflammatory cells in the lymph vessels in theconjunctiva, an elevation in the number of inflammatory cells present inthe lymph vessels of the conjunctiva, as compared to a reference levelof the one or more ocular physical parameters, can also be selected fortreatment (e.g., any of the treatments described herein). The methodscan also include recording the results of any of the methods describedherein (e.g., diagnosed as having an eye inflammation (e.g., diagnosedas having dry eye syndrome, limbal stem cell deficiency, allergy, orgraft versus host disease), or selected for treatment of eyeinflammation (e.g., selected for treating of dry eye syndrome, limbalstem cell deficiency, allergy, or graft versus host disease)) in thesubject's medical records (e.g., recording the results in a computerreadable medium), performing a diagnostic test for eye inflammation(e.g., dry eye syndrome, limbal stem cell deficiency, allergy, or graftversus host disease) on one or more lineal family members of a subjectdiagnosed as having eye inflammation (e.g., diagnosed as having dry eyesyndrome, limbal stem cell deficiency, allergy, or graft versus hostdisease) using the methods described herein, or monitoring one or morelineal family members of a subject diagnosed as having eye inflammation(e.g., diagnosed as having dry eye disorder, allergy, limbal stem cellinsufficiency, or graft versus host disease) using the methods describedherein for the development of eye inflammation (e.g., the development ofdry eye syndrome, limbal stem cell deficiency, allergy, or graft versushost disease) (e.g., using any of the methods described herein).

Dry Eye Syndrome

Dry eye syndrome is one of the most common eye problems affecting thegeneral population. A tear film normally provides the cornea andconjunctiva with protection from the air. Patients with dry eye syndromelack a tear film on some surfaces of the cornea and conjunctiva thatleads to symptoms of irritation and changing vision. Non-limitingexamples symptoms of dry eye syndrome include: stinging, burning, orscratchy sensation in the eyes, stringy mucus in or around the eyes,increased eye irritation from smoke or wind, eye fatigue after shortperiods of reading, sensitivity to light, periods of excessive tearing,and blurred vision (often worsening at the end of the day or afterfocusing for a prolonged period). Patients having dry eye syndrome arecommonly prescribed artificial tears, an oral antibiotic, and/orcyclosporine. Dry eye syndrome is also referred to as dry eye disease inthe art.

In some embodiments, a subject having dry eye syndrome does not have adetectable or observable amount of inflammation (e.g., a subject in alater stage of the disease). In some embodiments, a subject having dryeye syndrome can have an abnormality in the nerves in the cornea (e.g.,any of the nerve cell changes observed in subjects having dry eyesyndrome described herein) and abnormal epithelial cells in the cornea(e.g., any of the epithelial cell changes observed in subjects havingdry eye syndrome described herein), while showing no change in dendriticimmune cells in the cornea (e.g., compared to subjects not having eyedisorder (e.g., a subject not having dry eye syndrome).

A subject can be diagnosed as having dry eye syndrome by assessment ofone or more (e.g., two, three, four, five, six, seven, eight, nine, orten) of the above symptoms of dry eye syndrome in a subject. Anothertest that is used to diagnose dry eye syndrome is the Schirmer's teartest (e.g., a test which measures tear production using a blottingstrips of paper). Additional tests for diagnosing dry eye syndromeassess the quality of the subject's tears (e.g., the tear quality testcommercially available from TearLab Corporation). Additional tests thatcan, e.g., be performed to diagnose dry eye syndrome include cornealstaining (e.g., fluorescein staining) and conjunctival staining (e.g.,lissamine green or rose bengal staining)

Subjects can be diagnosed as having dry eye syndrome by a medicalprofessional (e.g., a physician, a physician's assistant, a nurse, anurse's assistant, or a laboratory technician). In any of the methodsdescribed herein, the subject can be a child, a teenager, or an adult(e.g., a subject at least 18, 25, 30, 40, 50, 60, 70, 80, or 90 yearsold). The subject can be a male or a female. A subject diagnosed ashaving dry eye syndrome may present with one or more (e.g., two, three,four, five, six, seven, eight, nine, and ten) of the symptoms of dry eyesyndrome described herein. In some embodiments, a subject having dry eyesyndrome may not present with a symptom of dry eye syndrome that can beeasily detected by basic examination of an eye(s) of the subject(examination of the patient that does not involve the magnification ofthe tissues of the eye). In some embodiments, the subject can bediagnosed as having dry eye syndrome based, in part, on the detection ofone or more (e.g., two, three, four, five, six, seven, eight, or nine)of the ocular physical parameters described herein. For example, thesubject can be diagnosed as having dry eye syndrome based on thedetection of one or more of the following ocular physical parameters(e.g., using in vivo confocal microscopy): an elevation in the number orpercentage of hyperreflective superficial epithelial cells present inthe cornea (e.g., in the center of the cornea), an elevation in theaverage size of superficial epithelial cells present in the cornea(e.g., in the center of the cornea), a decrease in the density ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), an elevation in the density of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), an elevationin the average size of dendritic immune cells present in the cornea(e.g., in the center of the cornea), an elevation in the average areacovered by dendritic immune cells present in the cornea (e.g., in thecenter of the cornea), a decrease in the density or average length ofnerves present in the cornea, a decrease in the amount of branching innerves present in the cornea, and a decrease in the total number ofnerves present in the cornea, as compared to a reference level of theone or more ocular physical parameters. The one or more referenceslevel(s) as described herein can be a threshold level (e.g., any of thethreshold values described herein) or can be a level of the one or moreocular physical parameters in a healthy subject (e.g., a subject thatdoes not have one or more symptoms of dry eye syndrome or an eyedisorder, or a subject that has not been diagnosed as having dry eyesyndrome or an eye disorder) or the same subject at an earlier timepoint (e.g., prior to onset of disease). The methods for determining theabove ocular physical parameters are described herein. A physician canalso monitor a subject for the development of dry eye syndrome byassessing one or more of these parameters at different time points overtime (e.g., at least once every six months, at least once a year, atleast once every two years, and at least once every three years).

A subject determined to have one or more (e.g., two, three, four, five,six, seven, eight, or nine) of: an elevation in the number or percentageof hyperreflective superficial epithelial cells present in the cornea(e.g., in the center of the cornea), an elevation in the average size ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), a decrease in the density of superficial epithelialcells present in the cornea (e.g., in the center of the cornea), anelevation in the density of dendritic immune cells present in the cornea(e.g., in the center of the cornea), an elevation in the average size ofdendritic immune cells present in the cornea (e.g., in the center of thecornea), an elevation in the average area covered by dendritic immunecells present in the cornea (e.g., in the center of the cornea), adecrease in the density or average length of nerves present in thecornea, a decrease in the amount of branching in nerves present in thecornea, and a decrease in the total number of nerves present in thecornea, as compared to a reference level of the one or more ocularphysical parameters, can also be selected for treatment (e.g., any ofthe treatments described herein).

In Vivo Microscopy

In vivo microscopy (e.g., in vivo confocal microscopy) is a noninvasiveprocedure that allows the imaging of the living cornea and conjunctivaat the cellular level. Additional non-invasive procedures can be used toperform the methods described herein. A non-invasive procedure, e.g., isone that does not require the puncturing or incision in the tissue ofthe subject (e.g., in the cornea of the subject).

In vivo microscopy is a technique that enables the study of cornealepithelial cells, corneal dendritic cells, corneal nerves, conjunctivalepithelial cells, conjunctival dendritic cells, conjunctival bloodvessels, and conjunctival lymphatic vessels. Exemplary methods fordetecting these specific cells and structures are described herein.

In vivo confocal microscopes are commercially available from, e.g.,Nidek Technologies (Gamagori, Japan) and Heidelberg Engineering GmbH(Dossenheim, Germany). In the methods described herein, the confocalmicroscopes are commonly equipped with an 35× to 70× immersion lens. Forexample, a Confoscan microscopy equipped with a 40×/0.75 objective lensor a Heidelberg Engineering GmbH microscope can be equipped with a 63×water-contact lens covered with a sterile single-usepolymethylmethacrylate cap (Tomocap, Heidelberg Engineering). TheConfoscan microscope, e.g., can produce images of 460 μm by 345 μm, witha magnification of 500× and a lateral resolution of 1 μm/pixel. TheHeidelberg microscope, e.g., can produce images of 400 μm by 400 μm,with a magnification of 800× and a lateral resolution of 1 μm/pixel. Thesubject is typically administered a topical anesthesia (e.g., 0.5%proparacaine hydrochloride) prior to contacting the immersion lens withthe subject's eye tissue. A subject can also be administered alubricating solution (e.g., 2.5% hydroxypropyl methylcellulose) prior tocontacting the immersion lens with the subject's eye tissue. The digitalimages collected can be stored on a computer workstation using commonlyknown methods. The resulting images can be analyzed using a variety ofcommercially available software. Non-limiting examples of software thatcan be used to analyze the collected images include ImageJ software(ImageJ software described in Girish et al., Indian J. Cancer 41:47,2004) and NeuronJ software (Meijering et al., Cytometry A 58:167-176,2004).

Changes in Superficial Epithelial Cells Present in the Cornea

As noted above, the present invention is based, in part, on thediscovery that efficacy of treatment of eye inflammation (e.g.,treatment of dry eye syndrome or limbal stem cell insufficiency) inhumans can be determined by detecting one or more of the change in thenumber or percentage of hyperreflective superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea), the change inthe average reflectivity of superficial epithelial cells present in thecornea (e.g., in the center of the cornea), the change in the averagesize of superficial epithelial cells present in the cornea (e.g., in thecenter of the cornea), and the change in the density of superficialepithelial cells present in the cornea (e.g., in the center of thecornea), where one or more of a decrease in the number or percentage ofhyperreflective superficial epithelial cells present in the cornea(e.g., in the center of the cornea), a decrease in the average size ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), and an elevation in the density of superficialepithelial cells present in the cornea (e.g., in the center of thecornea) following treatment or at a later time point during treatment ascompared to an earlier time point (e.g., a time point prior to treatmentor at an earlier time point during treatment) in the same subjectindicate that the treatment is effective.

Exemplary in vivo confocal microscopic methods for determining thesechanges in superficial epithelial cells present in the cornea aredescribed in the Examples. However, the methods described in theExamples are not limiting. One skilled in the art will recognize thatmodifications of these methods can be made (e.g., change in the level ofmagnification, change in autobrightness, the use of gel or the type ofcaps for the microscope objective lens) without significantlycompromising the quality of the images obtained. In some embodiments,two or more images (e.g., three, four, or five images) can be obtainedfrom an eye of the subject. A change in the number or percentage ofhyperreflective superficial epithelial cells or the average reflectivityof superficial epithelial cells present in the cornea can be assessedusing methods known in the art, e.g., the ImageJ, NIDEK, and Cell Count,Heidelberg Engineering GmbH software. A change in the average size ordensity of superficial epithelial cells present in the center of thecornea can be assessed using methods known in the art, e.g., the ImageJ,NIDEK, and Cell Count, Heidelberg Engineering GmbH software.

Changes in Dendritic Immune Cells Present in the Central and PeripheralCornea

The present invention is further based, in part, on the discovery thatefficacy of treatment of eye inflammation (e.g., treatment of dry eyesyndrome or limbal stem cell insufficiency) in humans can be determinedby detecting one or more of a change in the density of dendritic immunecells present in the cornea (e.g., in the center of the cornea), achange in the average size of dendritic immune cells present in thecornea (e.g., in the center of the cornea), and a change in the averagearea covered by dendritic immune cells present in the cornea (e.g., inthe center of the cornea), where one or more of a decrease in thedensity of dendritic immune cells present in the cornea (e.g., in thecenter of the cornea), a decrease in the average size of dendriticimmune cells present in the cornea (e.g., in the center of the cornea),and a decrease in the average area covered by dendritic immune cellspresent in the cornea (e.g., in the center of the cornea) followingtreatment or at a later time point during treatment as compared to anearlier time point (e.g., a time point prior to treatment or at anearlier time point during treatment) in the same subject indicate thatthe treatment is effective.

Exemplary methods using in vivo confocal microscopy for determiningthese changes in the dendritic immune cells present in the cornea aredescribed in the Examples. However, the methods described in theExamples are not limiting. One skilled in the art will recognize thatmodifications of these methods can be made (e.g., change in the level ofmagnification) without significantly compromising the quality of theimages obtained.

Dendritic cells can be morphologically identified as bright individualtree-like structures with cell bodies, which allows for thedifferentiation of these structures from the corneal nerves. The numberof dendritic cells present in the cornea can be counted using software(e.g., Cell Count, Heidelberg Engineering GmbH). The average dendriticcell size and average dendritic cell area can also be analyzed usingsoftware (e.g., ImageJ software described in Girish et al., Indian J.Cancer 41:47, 2004). Although exemplary software programs are recitedabove, skilled artisans will appreciate that a number of other suitablesoftware programs are available.

Subjects having acute allergy have also been discovered to have anelevation in dendritic cell density in the peripheral cornea, anelevation in the average dendritic cell size in the peripheral cornea,and an elevation in the number or percentage of hyperreflectivedendritic cells in the peripheral cornea. As described herein, thedetection of one or more of a change in the dendritic cell density inthe peripheral cornea, a change in the average dendritic cell size, achange in the number or percentage of hypperreflective dendritic cellsin the peripheral cornea in a subject can be used to select a treatmentfor a subject having eye inflammation (e.g., a subject having dry eyesyndrome, limbal stem cell insufficiency, allergy, or graft versus hostdisease).

Changes in Nerve Cells in the Cornea

It has further been discovered that efficacy of treatment of eyeinflammation (e.g., treatment of dry eye syndrome or limbal stem cellinsufficiency) in humans can be determined by detecting one or more of achange in the density or average length of nerves present in the cornea,a change in the amount of branching in nerves present in the cornea, anda change in the total number of nerves present in the cornea, where oneor more of an elevation in the density or average length of nervespresent in the cornea, an elevation in the amount of branching in nervespresent in the cornea, and an elevation in the total number of nervespresent in the cornea following treatment or at a later time pointduring treatment as compared to an earlier time point (e.g., a timepoint prior to treatment or at an earlier time point during treatment)in the same subject indicate that the treatment is effective.

Exemplary in vivo confocal microscopic methods for determining thesechanges in the nerve cells present in the cornea are described in theExamples. However, the methods described in the Examples are notlimiting. One skilled in the art will recognize that modifications ofthese methods can be made (e.g., change in the level of magnification)without significantly compromising the quality of the images obtained.

Nerve analysis can be done, e.g., using a software program (e.g., thesemi-automated tracing program NeuronJ (Meijering et al., Cytometry A58:167-176, 2004), a plug-in for ImageJ (ImageJ software described inGirish et al., Indian J. Cancer 41:47, 2004)). In some embodiments,nerve density can be assessed by measuring the total length of the nervefibers in micrometers per frame. Nerve branching is defined as the totalnumber of nerve branches in one image. The number of total nervesmeasured is defined as the number of all nerves, including main nervetrunks and branches in one image. Although exemplary software programsare recited above, skilled artisans will appreciate that a number ofother suitable software programs are available.

Determining Efficacy of a Treatment for Dry Eye Syndrome

Provided herein are methods of determining the efficacy of a treatmentfor eye inflammation (e.g., treatment for dry eye syndrome or limbalstem cell insufficiency) in a subject. These methods include determiningin an eye of a subject having eye inflammation (e.g., a subject havingdry eye syndrome or limbal stem cell insufficiency) one or more (e.g.,two, three, four, five, six, seven, eight, or nine) of (i) the number orpercentage of hyperreflective superficial epithelial cells present inthe cornea (e.g., in the center of the cornea), (ii) the average size ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), (iii) the density of superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea), (iv) thedensity of dendritic immune cells present in the cornea (e.g., in thecenter of the cornea), (v) the average size of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), (vi) theaverage area covered by dendritic immune cells present in the cornea(e.g., in the center of the cornea), (vii) the density or average lengthof nerves present in the cornea, (viii) the amount of branching innerves present in the cornea, and (ix) the total number of nervespresent in the cornea at a first time point; determining one or more of(i) the number or percentage of hyperreflective superficial epithelialcells present in the cornea (e.g., in the center of the cornea), (ii)the average size of superficial epithelial cells present in the cornea(e.g., in the center of the cornea), (iii) the density of superficialepithelial cells present in the cornea (e.g., in the center of thecornea), (iv) the density of dendritic immune cells present in thecornea (e.g., in the center of the cornea), (v) the average size ofdendritic immune cells present in the cornea (e.g., in the center of thecornea), (vi) the average area covered by dendritic immune cells presentin the cornea (e.g., in the center of the cornea), (vii) the density oraverage length of nerves present in the cornea, (viii) the amount ofbranching in nerves present in the cornea, and (ix) the total number ofnerves present in the cornea in the eye of the subject at a second timepoint; and comparing the one or more of (i)-(ix) determined at the firstand second time points, where the first time point is prior to treatmentand the second time point is any time point following the initiation oftreatment, or the first time point is following the initiation oftreatment and the second time point is at a later time point during orafter treatment; and one or more (e.g., two, three, four, five, six,seven, eight, or nine) of a decrease in the number or percentage ofhyperreflective superficial epithelial cells present in the cornea(e.g., in the center of the cornea), a decrease in the average size ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), an elevation in the density of superficial epithelialcells present in the cornea (e.g., in the center of the cornea), adecrease in the density of dendritic immune cells present in the cornea(e.g., in the center of the cornea), a decrease in the average size ofdendritic immune cells present in the cornea (e.g., in the center of thecornea), and a decrease in the average area covered by dendritic immunecells present in the center of the cornea, an elevation in the densityor average length of nerves present in the cornea, an elevation in theamount of branching in nerves present in the cornea, and an elevation inthe total number of nerves present in the cornea, determined at thesecond time point compared to the first time point indicates that thetreatment was effective in the subject. In some embodiments, thedetermining is performed using in vivo confocal microscopy.

Alternatively, in the above methods, a subject that has one or more(e.g., two, three, four, five, six, seven, eight, or nine) of anelevation or no substantial change in the number or percentage ofhyperreflective superficial epithelial cells present in the cornea(e.g., in the center of the cornea), an elevation or no substantialchange in the average size of superficial epithelial cells present inthe cornea (e.g., in the center of the cornea), a decrease or nosubstantial change in the density of superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea), an elevationor no substantial change in the density of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), an elevationor no substantial change in the average size of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), and anelevation in the average area covered by dendritic immune cells presentin the cornea (e.g., in the center of the cornea), a decrease or nosubstantial change in the density or average length of nerves present inthe cornea, a decrease or no substantial change in the amount ofbranching in nerves present in the cornea, and a decrease or nosubstantial change in the total number of nerves present in the cornea,determined at the second time point compared to the first time pointindicates that the treatment was not effective in the subject. Someembodiments, where the treatment has been indicated to be ineffective inthe subject, further include administering, recommending, or prescribingan alternate treatment to the subject. In some embodiments, thealternate treatment can be a different therapeutic agent or a differentcombination of one or more therapeutic agents. In some embodiments, thealternate treatment can be an increased dosage of one or moretherapeutic agents currently being taken by the subject, an increase inthe frequency of administration of one or more therapeutic agentscurrently being taken by the subject, or an alteration in the route ofdelivery of one or more therapeutic agents being currently taken by thesubject. Some embodiments further include recording the results of thesemethods in the subject's medical records (e.g., recording the results ina computer readable medium), performing a diagnostic test for eyeinflammation (e.g., dry eye syndrome, limbal stem cell deficiency,allergy, or graft versus host disease) on one or more lineal familymembers of the subject using the methods described herein, or monitoringone or more lineal family members of the subject diagnosed using themethods described herein for the development of eye inflammation (e.g.,the development of dry eye syndrome, limbal stem cell deficiency,allergy, or graft versus host disease) (e.g., using any of the methodsdescribed herein).

Some embodiments further include assessing of one or more (e.g., two,three, four, or five) additional symptoms of eye inflammation (e.g., oneor more symptoms of dry eye syndrome) in the subject at the first and/orsecond time point (e.g., additional symptoms of dry eye syndrome thatcan be assessed without the use of a microscope). Some embodimentsfurther include performing a tear production test (e.g., Schirmer'stest) or a tear quality test (e.g., TearLab® Osmolarity Test, TearLabCorporation) at the first and/or second time point. In some embodiments,the methods are performed as part of a clinical trial or are performedas part of a treatment regimen.

In some embodiments, these methods can be performed by a medicalprofessional (e.g., a physician, a physician's assistant, a nurse, anurse's assistant, or a laboratory technician). In some embodiments, thesubject has been diagnosed as having eye inflammation (e.g., diagnosedas having dry eye syndrome, limbal stem cell insufficiency, graft versushost disease, or allergy). In some embodiments, the subject has anincreased risk of developing eye inflammation (e.g., increased risk ofdeveloping dry eye syndrome, limbal stem cell insufficiency, or graftversus host disease, allergy) or is suspected of having eye inflammation(e.g., suspected of having dry eye syndrome, limbal stem cellinsufficiency, allergy, or graft versus host disease). In someembodiments, the subject has one or more symptoms of eye inflammation(e.g., one or more symptoms of dry eye syndrome, limbal stem cellinsufficiency, graft versus host disease, or allergy). In someembodiments, the subject does not present with a symptom of eyeinflammation (e.g., a symptom of dry eye syndrome, limbal stem cellinsufficiency, allergy, or graft versus host disease) that can beobserved without the use of a microscope. In some embodiments, thesubject has a form of eye inflammation (e.g., a form of dry eyesyndrome, limbal stem cell insufficiency, graft versus host disease, orallergy) that is refractory to previous therapeutic treatment. In someembodiments, the subject has had eye inflammation (e.g., dry eyesyndrome, limbal stem cell insufficiency, allergy, or graft versus hostdisease) for at least one week (e.g., at least two weeks, three weeks,one month, two months, three months, four months, six months, or oneyear). In some embodiments, the eye inflammation (e.g., dry eyesyndrome) can be caused by an autoimmune condition (e.g., any of theautoimmune conditions described herein).

In some embodiments, the subject has a form of dry eye syndrome withboth an evaporative and aqueous deficient component. In some embodiment,the subject has aqueous deficient form of dry eye syndrome (e.g., causedby Sjogren's syndrome or graft-versus-host disease). An aqueousdeficient form of dry eye syndrome implies that a failure of lacrimaltear secretion is the cause of symptoms and ocular changes in thesubject, and can be, e.g., caused by lacrimal acinar destruction ordysfunction. In some embodiments, the subject has an evaporative form ofdry eye syndrome. An evaporative form of dry eye syndrome ischaracterized by, e.g., an excessive water loss from an exposed ocularsurface in the presence of normal lacrimal secretory function.

In some embodiments, the subject is a child, a teenager, or an adult(e.g., at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,or 90 years old). In some embodiments, the subject is a female (e.g., apost-menopausal female). In some embodiments, the subject is a male. Insome embodiments, the subject is already receiving a treatment for eyeinflammation (e.g., a treatment for dry eye syndrome, limbal stem cellinsufficiency, graft versus host disease, or allergy), the subjectterminates the previous treatment for eye inflammation (e.g., treatmentfor dry eye syndrome, limbal stem cell insufficiency, graft versus hostdisease, or allergy), and the efficacy of a new treatment is determinedusing the methods described herein. In some embodiments, the subject isalready receiving a treatment for eye inflammation (e.g., treatment fordry eye syndrome, limbal stem cell insufficiency, graft versus hostdisease, or allergy), the subject begins to take one or more additional(new) therapeutic agent(s) in combination with the old treatment, andthe efficacy of the combination of the one or more additional (new)therapeutic agents and the old treatment are determined using themethods described above. In some embodiments, the subject is alreadyreceiving a one or more therapeutic agent(s) for eye inflammation (e.g.,one or more therapeutic agent(s) for dry eye syndrome, limbal stem cellinsufficiency, allergy, or graft versus host disease), and the efficacyof an increased dosage and/or an increased frequency of dosing of thepreviously administered one or more therapeutic agent(s) is determinedusing the methods described herein. In some embodiments, the subject isalready receiving one or more therapeutic agent(s) for eye inflammation(e.g., one or more therapeutic agent(s) for dry eye syndrome, limbalstem cell insufficiency, graft versus host disease, or allergy), and theefficacy of an alternative route of administration of the one or moretherapeutic agent(s) previously administered to the subject isdetermined using the methods described above.

In some embodiments, the amount of time between the first and the secondtime point can be at least one week (e.g., at least two weeks, threeweeks, one month, two months, three months, four months, six months, orone year).

Some embodiments further include administering a treatment (e.g., one ormore therapeutic agents) to the subject between the first and secondtime points. Some embodiments further include administering a treatmentto the subject prior to the first time point. Some embodiments furtherinclude determining one or more (e.g., two, three, four, five, six,seven, eight, or nine) of (i)-(ix) at one or more additional time points(e.g., after the second time point) in the eye of the subject having eyeinflammation (e.g., a subject having dry eye syndrome, limbal stem cellinsufficiency, graft versus host disease, or allergy). In someembodiments, the one or more additional time points occur after the endof the therapeutic treatment.

Methods of Treating a Subject

Also provided are methods of treating a subject having eye inflammation(e.g., a subject having dry eye syndrome, limbal stem cellinsufficiency, graft versus host disease, or allergy) that includeselectively administering to an eye(s) of a subject having eyeinflammation (e.g., subject having dry eye syndrome, limbal stem cellinsufficiency, graft versus host disease, or allergy) and determined tohave an elevated density of dendritic immune cells present in the centerof the cornea as compared to a reference level, a topical steroidsolution; or selectively orally or topically administering to a subjecthaving eye inflammation (e.g., a subject having dry eye syndrome, limbalstem cell insufficiency, graft versus host disease, or allergy) anddetermined to have substantially no change or a decreased density ofdendritic immune cells present in the center of the cornea as comparedto a reference level, at least two (e.g., three, or four)immunosuppressive agents (e.g., at least one steroid).

Some embodiments further include one or more of: determining a level ofdendritic immune cells present in the center of the cornea of thesubject; comparing the level of dendritic immune cells present in thecenter of the cornea of the subject to a reference level; and selectinga subject that has an elevated level of dendritic immune cells presentin the center of the cornea as compared to the reference level fortreatment. Some embodiments further include selecting a subject havingeye inflammation (e.g., a subject having dry eye syndrome, limbal stemcell insufficiency, graft versus host disease, or allergy). In someembodiments, the determining is performed using in vivo confocalmicroscopy.

In some embodiments, these methods are performed by a medicalprofessional (e.g., a physician, a physician's assistant, a nurse, anurse's assistant, or a laboratory technician). In some embodiments, thesubject may already be taking one or more pharmaceutical agents fortreatment of eye inflammation (e.g., one or more pharmaceutical agentsfor treatment of dry eye syndrome, graft versus host disease, limbalstem cell insufficiency, or allergy), and the subject is instructed oradvised to discontinue taking one or more of the previously prescribedone or more pharmaceutical agents. In some embodiments, the subject mayalready be taking one or more pharmaceutical agents for treatment of eyeinflammation (e.g., one or more pharmaceutical agents for treatment ofdry eye syndrome, limbal stem cell insufficiency, allergy, or graftversus host disease), and the topical steroid solution or the topically-or orally-administered at least two (e.g., three or four)immunosuppressive agents is administered to the subject in combinationwith the one or more pharmaceutical agents previously taken by thesubject.

In some embodiments, the reference level can be a threshold level or canbe the density of dendritic immune cells present in the center of thecornea in a healthy subject (e.g., a subject that does not have an eyedisorder, or does not have one or more symptoms of an eye disorder(e.g., an inflammatory eye disease such as dry eye syndrome, acuteallergy, and chronic allergy) or a subject that has not been diagnosedas having an eye disorder (e.g., an inflammatory eye disease such as dryeye syndrome, acute allergy, or chronic allergy)) or the same subject atan earlier time point.

In some embodiments, the topical steroid solution contains one or more(e.g., two, three, or four) steroid(s) selected from the group ofloteprednol etabonate, cortisone, dexamethasone, hydrocortisone,prednisolone, prednisone, methylprednisolone, betamethasone,dexamethasone, rimexolone, prednisolone acetate, triamcinolone,beclometasone, fludrocortisone, deoxycorticosterone, aldosterone,betamethasone, deoxycorticosterone, and aldosterone. In someembodiments, the topical steroid solution comprises loteprednoletabonate. In some embodiments, the topical steroid solution comprises1% or 0.12% prednisolone acetate. Additional examples of topical steroidsolutions are known in the art. In some embodiments, the topical steroidsolution is administered to an eye of the subject at least once a day(e.g., at least twice, three times, or four times a day). In someembodiments, the administration of the topical steroid solution occursbefore bedtime and/or shortly after awakening (e.g., within one hour ofawakening) in the morning. Additional non-limiting methods of treating asubject having dry eye syndrome are shown in FIG. 15.

In some embodiments, the at least two (e.g., three or four)immunosuppressive agents that is topically administered is selected fromthe group of antibodies (e.g., fully human or humanized antibodies) thatspecifically bind to CD20, CD25 (e.g., basiliximab or daclizumab), orCD3 (e.g., muromonab); calcineurin inhibitors (e.g., ciclosporin,pimecrolimus, tacrolimus, sirolimus, and/or cyclosporine); interferons(e.g., interferon-β); steroids (e.g., any of the steroids known in theart or described herein); interleukin-1 receptor antagonists;myophenolate mofetil; Prograph®; azathioprine; methotrexate; and/orTNF-α binding proteins (e.g., antibodies and/or soluble TNF-α receptors,e.g., infliximab, etanercept, and/or adalimumab). Additional examples ofimmunosuppressive agents that can be topically administered are known inthe art.

In some embodiments, the at least one of the at least twoimmunosuppressive agents is a steroid (e.g., one or more of loteprednoletabonate, cortisone, dexamethasone, hydrocortisone, prednisolone,prednisone, methylprednisolone, betamethasone, dexamethasone,triamcinolone, beclometasone, fludrocortisone, deoxycorticosterone,aldosterone, betamethasone, deoxycorticosterone, and/or aldosterone). Insome embodiments, at least one of the at least two immunosuppressiveagents is a steroid and at least one of the at least twoimmunosuppressive agents is selected from pimecrolimus, tacrolimus,sirolimus, and cyclosporine. Additional examples of immunosuppressiveagents that can be used in these methods are known in the art.

In some embodiments, the at least two (e.g., three or four)immunosuppressive agents are topically and/or orally administered to aneye of the subject at least twice a week (e.g., at least twice, threetimes, or four times a day). In some embodiments, the topical and/ororal administration of the at least two immunosuppressive agents occursbefore bedtime and/or shortly after awakening (e.g., within one hour ofawakening) in the morning. In some embodiments, the at least twoimmunosuppressive agents are formulated together (e.g., present in thesame liquid formulation for optical administration). In someembodiments, the at least two immunosuppressive agents are formulated asseparate compositions (e.g., each formulated in separate liquidformulations for optical administration).

Also provided are methods of treating a subject (e.g., a subject havingeye inflammation, e.g., a subject having dry eye syndrome, acuteallergy, chronic allergy, limbal stem cell insufficiency, or graftversus host disease, or any of the other inflammatory eye diseasesdescribed herein) that include selectively orally or topicallyadministering to a subject, determined to have an elevated number orelevated average density of dendritic inflammatory cells present in theperipheral cornea as compared to a corresponding reference value, atleast one (e.g., two, three, or four) anti-inflammatory steroid and/orat least one (e.g., two, three, or four) immunosuppressive agent (e.g.,at least one calcineurin inhibitor).

Some embodiments further include one or more of: determining the levelof the number or average density of dendritic inflammatory cells presentin the peripheral cornea; comparing level of the number or averagedensity of dendritic inflammatory cells present in the peripheral corneaof the subject to a reference level; and selecting a subject having anelevated number or average density of dendritic inflammatory cellspresent in the peripheral cornea as compared to the reference level fortreatment. In some embodiments, the determining is performed using invivo confocal microscopy. Some embodiments further include selecting asubject having an inflammatory eye disease (e.g., dry eye syndrome,acute allergy, chronic allergy, or any of the other inflammatory eyediseases described herein).

In some embodiments, these methods are performed by a medicalprofessional (e.g., a physician, a physician's assistant, a nurse, anurse's assistant, or a laboratory technician). In some embodiments, thesubject may already be taking one or more pharmaceutical agents fortreatment of inflammatory eye disorder, and the subject is instructed oradvised to discontinue taking one or more of the previously prescribedone or more pharmaceutical agents. In some embodiments, the subject mayalready be taking one or more pharmaceutical agents for treatment of aninflammatory eye disorder, and the at least one anti-inflammatorysteroid and/or the at least one immunosuppressive agent (e.g., at leastone calcineurin inhibitor) is administered to the subject in combinationwith the one or more pharmaceutical agents previously taken by thesubject.

In some embodiments, the reference level can be a threshold level or canbe number or average density of dendritic inflammatory cells present inthe peripheral cornea in a healthy subject (e.g., a subject that doesnot have one or more symptoms of eye disease (e.g., an inflammatory eyedisease such as dry eye syndrome, acute allergy, or chronic allergy) ora subject that has not been diagnosed as having an eye disease (e.g., aninflammatory eye disease such as dry eye syndrome, acute allergy, orchronic allergy)) or the same subject at an earlier time point.

In some embodiments, the at least one anti-inflammatory steroid isselected from the group of: hydrocortisone, cortisone, prednisone,prednisolone, methylprednisolone, dexamethasone, betamethasone,triamcinolone, beclometasone, fludrocortisone, deoxycorticosterone, andaldosterone. Additional examples of anti-inflammatory steroids that can,e.g., be used in the methods described herein are known in the art. Insome embodiments, the at least one anti-inflammatory steroid isadministered to an eye of the subject at least once a day (e.g., atleast twice, three times, four times a day). In some embodiments, the atleast one anti-inflammatory steroid is orally administered to thesubject at least once a week (e.g., at least twice a week or at leastonce a day). In some embodiments, the at least one anti-inflammatorysteroid occurs before bedtime and/or shortly after awakening (e.g.,within one hour of awakening) in the morning. In some embodiments, theat least one anti-inflammatory steroid and the at least oneimmunosuppressive agent (e.g., a calcineurin inhibitor) are administeredat approximately the same time (e.g., within 10 minutes of each other).

In some embodiments, the at least one (e.g., two, three, or four)immunosuppressive agent is a calcineurin inhibitor (e.g., ciclosporin,pimecrolimus, tacrolimus, sirolimus, or cyclosporine). Additionalexamples of immunosuppressive agents that can be used in the methodsdescribed herein are known in the art and are described herein (e.g.,any of the immunosuppressive agents described herein). In someembodiments, the at least one (e.g., two, three, or four)immunosuppressive agent is topically administered to an eye of thesubject at least once a day (e.g., at least twice, three times, fourtimes a day). In some embodiments, the at least one immunosuppressiveagent is orally administered to the subject at least once a week (e.g.,at least twice a week or at least once a day). In some embodiments, thetopical administration of the at least one anti-inflammatory steroidand/or the at least one immunosuppressive agent occurs before bedtimeand/or shortly after awakening (e.g., within one hour of awakening) inthe morning. In some embodiments, the at least one anti-inflammatoryagent and the at least one immunosuppressive agent are present in thesame formulation (e.g., a pharmaceutically acceptable solution foroptical administration or a solid formulation (e.g., a pill or capsule)for oral administration).

Selecting a Subject for Participation in a Clinical Study

Also provided are methods of selecting a subject (e.g., a subject havingeye inflammation, e.g., dry eye syndrome, acute allergy, chronicallergy, limbal stem cell insufficiency, or graft versus host disease)for participation in a clinical study. These methods include determininga level of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, or 19) of the following ocular physical parameters:(i) the number or percentage of hyperreflective superficial epithelialcells present in the cornea (e.g., in the center of the cornea), (ii)the average size of superficial epithelial cells present in the cornea(e.g., in the center of the cornea), (iii) the density of superficialepithelial cells present in the cornea (e.g., in the center of thecornea), (iv) the density of dendritic immune cells present in thecornea (e.g., in the center of the cornea), (v) the average size ofdendritic immune cells present in the cornea (e.g., in the center of thecornea), (vi) the average area covered by dendritic immune cells presentin the cornea (e.g., in the center of the cornea), (vii) the density oraverage length of nerves present in the cornea, (viii) the amount ofbranching in nerves present in the cornea, (ix) the total number ofnerves present in the cornea, (x) the number or average density ofdendritic inflammatory cells present in the peripheral cornea, (xi) thehyperreflectivity of epithelial cells in the conjunctiva, (xii) thedendritic cell density in the conjunctiva, (xiii) the average dendriticcell size in the conjunctiva, (xiv) the number of hyperreflectivedendritic cells in the conjunctiva, (xv) the dilation of the lumen ofblood vessels in the conjunctiva, (xvi) the average size of inflammatorycells in the blood vessels in the conjunctiva, (xvii) the sticking (theaverage time of transitory residence) of inflammatory cells to the bloodvessels walls in the conjunctiva, (xviii) the average size ofinflammatory cells in the lymph vessels in the conjunctiva, and (xix)the number of inflammatory cells present in the lymph vessels of theconjunctiva, as compared to a reference level of the one or more ocularphysical parameters; comparing the one or more of (i)-(xix) (listedabove) determined in the eye of the subject to one or more correspondingreference values; and selecting a subject having one or more (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of: anelevation in the number or percentage of hyperreflective superficialepithelial cells present in the cornea (e.g., in the center of thecornea), an elevation in the average size of superficial epithelialcells present in the cornea (e.g., in the center of the cornea), adecrease in the density of superficial epithelial cells present in thecornea (e.g., in the center of the cornea), an elevation in the densityof dendritic immune cells present in the cornea (e.g., in the center ofthe cornea), an elevation in the average size of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), an elevationin the average area covered by dendritic immune cells present in thecornea (e.g., in the center of the cornea), a decrease in the density oraverage length of nerves present in the cornea, a decrease in the amountof branching in nerves present in the cornea, a decrease in the totalnumber of nerves present in the cornea, an elevation in the number oraverage density of dendritic inflammatory cells present in theperipheral cornea, an elevation in the reflectivity of epithelial cellsin the conjunctiva, an elevation in dendritic cell density in theconjunctiva, an elevation in the average dendritic cell size in theconjunctiva, an elevation in the number of hyperreflective dendriticcells in the conjunctiva, an elevation in the dilation of the lumen ofblood vessels in the conjunctiva, an elevation in the average size ofinflammatory cells in the blood vessels in the conjunctiva, an elevationin the sticking (elevation in the average time of transitory residence)of inflammatory cells to the blood vessels walls in the conjunctiva, anelevation in the average size of inflammatory cells in the lymph vesselsin the conjunctiva, an elevation in the number of inflammatory cellspresent in the lymph vessels of the conjunctiva, compared to the one ormore corresponding reference values for participation in a clinicalstudy. In some embodiments, the determining is performed using in vivoconfocal microscopy.

Alternatively, in the above methods, a subject having one or more (e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of adecrease or no substantial difference in the number or percentage ofhyperreflective superficial epithelial cells present in the cornea(e.g., in the center of the cornea), a decrease or no substantialdifference in the average size of superficial epithelial cells presentin the cornea (e.g., in the center of the cornea), an elevation or nosubstantial change in the density of superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea), a decrease orno substantial change in the density of dendritic immune cells presentin the cornea (e.g., in the center of the cornea), a decrease or nosubstantial change in the average size of dendritic immune cells presentin the cornea (e.g., in the center of the cornea), a decrease or nosubstantial change in the average area covered by dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), an elevationor no substantial change in the density or average length of nervespresent in the cornea, an elevation or no substantial change in theamount of branching in nerves present in the cornea, an elevation or nosubstantial change in the total number of nerves present in the cornea,a decrease or no substantial change in the number or average density ofdendritic inflammatory cells present in the peripheral cornea, adecrease or no substantial change in the hyperreflectivity of epithelialcells in the conjunctiva, a decrease or no substantial change indendritic cell density in the conjunctiva, a decrease or no substantialchange in the average dendritic cell size in the conjunctiva, a decreaseor no substantial change in the number of hyperreflective dendriticcells in the conjunctiva, a decrease or no substantial change in thedilation of the lumen of blood vessels in the conjunctiva, a decrease orno substantial change in the average size of inflammatory cells in theblood vessels in the conjunctiva, a decrease or no substantial change inthe sticking (a decrease or no substantial change in the average time oftransitory residence) of inflammatory cells to the blood vessels wallsin the conjunctiva, a decrease or no substantial change in the averagesize of inflammatory cells in the lymph vessels in the conjunctiva, adecrease or no substantial change in the number of inflammatory cellspresent in the lymph vessels of the conjunctiva, compared to the one ormore corresponding reference values is selected for participation in aclinical study (e.g., selected as a control subject).

Also provided are methods of selecting a subject (e.g., a subject havingdry eye syndrome, limbal stem cell insufficiency, allergy, or graftversus host disease) for participation in a clinical study that include:determining a level of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) ofthe following ocular physical parameters: (i) the number or percentageof hyperreflective superficial epithelial cells present in the cornea(e.g., in the center of the cornea), (ii) the average size ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), (iii) the density of superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea), (iv) thedensity of dendritic immune cells present in the cornea (e.g., in thecenter of the cornea), (v) the average size of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), (vi) theaverage area covered by dendritic immune cells present in the cornea(e.g., in the center of the cornea), (vii) the density or average lengthof nerves present in the cornea, (viii) the amount of branching innerves present in the cornea, and (ix) the total number of nervespresent in the cornea; comparing the one or more of (i) the number orpercentage of hyperreflective superficial epithelial cells present inthe cornea (e.g., in the center of the cornea), (ii) the average size ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), (iii) the density of superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea), (iv) thedensity of dendritic immune cells present in the cornea (e.g., in thecenter of the cornea), (v) the average size of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), (vi) theaverage area covered by dendritic immune cells present in the cornea(e.g., in the center of the cornea), (vii) the density or average lengthof nerves present in the cornea, (viii) the amount of branching innerves present in the cornea, and (ix) the total number of nervespresent in the cornea, determined in the eye of the subject to one ormore corresponding reference values; and selecting a subject having oneor more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) of an elevation in the numberor percentage of hyperreflective superficial epithelial cells present inthe cornea (e.g., in the center of the cornea), an elevation in theaverage size of superficial epithelial cells present in the cornea(e.g., in the center of the cornea), a decrease in the density ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), an elevation in the density of dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), an elevationin the average size of dendritic immune cells present in the cornea(e.g., in the center of the cornea), an elevation in the average areacovered by dendritic immune cells present in the cornea (e.g., in thecenter of the cornea), a decrease in the density or average length ofnerves present in the cornea, a decrease in the amount of branching innerves present in the cornea, a decrease in the total number of nervespresent in the cornea, compared to the one or more correspondingreference values for participation in a clinical study. In someembodiments, the determining is performed using in vivo confocalmicroscopy.

Alternatively, in the above methods, a subject having one or more (e.g.,2, 3, 4, 5, 6, 7, 8, or 9) of an elevation or no substantial change inthe number or percentage of hyperreflective superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea), a decrease orno substantial change in the average size of superficial epithelialcells present in the cornea (e.g., in the center of the cornea), anelevation or no substantial change in the density of superficialepithelial cells present in the cornea (e.g., in the center of thecornea), a decrease or no substantial change in the density of dendriticimmune cells present in the cornea (e.g., in the center of the cornea),a decrease or no substantial change in the average size of dendriticimmune cells present in the cornea (e.g., in the center of the cornea),a decrease or no substantial change in the average area covered bydendritic immune cells present in the cornea (e.g., in the center of thecornea), an elevation or no substantial change in the density or averagelength of nerves present in the cornea, an elevation or no substantialchange in the amount of branching in nerves present in the cornea, anelevation or no substantial change in the total number of nerves presentin the cornea, compared to the one or more corresponding referencevalues is selected for participation in a clinical study (e.g., selectedas a control subject).

Also provided are methods for selecting a subject (e.g., a subjecthaving eye inflammation, e.g., acute or chronic allergy, dry eyesyndrome, limbal stem cell insufficiency, or graft versus host disease)for participation in a clinical study that include: determining in aneye of the subject the number or average density of dendriticinflammatory cells present in the peripheral cornea; comparing thenumber or average density of dendritic inflammatory cells in theperipheral cornea in the eye of the subject to a corresponding referencevalue; and selecting a subject having an elevation in the number oraverage density of dendritic inflammatory cells compared to thecorresponding reference value for participation in a clinical study.Alternatively, a subject that has a decrease or no substantial change inthe number or average density of dendritic inflammatory cells in theperipheral cornea compared to the corresponding reference value isselected for participation in a clinical study (e.g., as a controlsubject).

In some embodiments of the methods described herein, the subject hasbeen diagnosed with dry eye syndrome or another inflammatory eyedisorder (e.g., any of the inflammatory eye disorders described herein,e.g., acute or chronic allergy, limbal stem cell insufficiency, or graftversus host disease). In some embodiments of the methods describedherein, the subject has not been diagnosed as having dry eye syndrome oranother inflammatory eye disorder (e.g., any of the inflammatory eyedisorders described herein). In some embodiments of the methodsdescribed herein, the subject is at increased risk of developing dry eyesyndrome or another inflammatory eye disorder (e.g., any of theinflammatory eye disorders described herein) or is suspected of havingdry eye syndrome or another inflammatory eye disorder described herein.In some embodiments, the subject has at least one symptoms of dry eyesyndrome or another inflammatory eye disorder (e.g., any of theinflammatory eye disorders described herein).

In some embodiments, the reference level can be a threshold level (e.g.,any of the threshold values described herein) or can be a level of theone or more ocular physical parameters in a healthy subject (e.g., asubject that does not have one or more symptoms of an eye disorder(e.g., an inflammatory eye disease such as dry eye syndrome, acuteallergy, or chronic allergy) or a subject that has not been diagnosed ashaving an eye disorder (e.g., an inflammatory eye disease such as dryeye syndrome, acute allergy, or chronic allergy)) or the same subject atan earlier time point.

The invention is further described in the following example, which doesnot limit the scope of the invention described in the claims.

Examples Example 1. Detection of Changes in the Superficial EpithelialCells Present in the Central Cornea of Patients Having Dry Eye SyndromeUpon Treatment

In vivo confocal microscopy was performed to image the superficialepithelial cells present in the central corneas of three subjects havingdry eye syndrome (i) before or at an early time point in therapy, and(ii) at a second later time point. Each of these patients demonstrated agood response to treatment for his or her dry eye syndrome.

Images were gathered from subject #1 prior to treatment and at 6-weeksafter the initiation of treatment (topical loteprednol four times a dayfor four weeks, followed by twice daily administration of topicalloteprednol, with daily administration of artificial tears and RefreshPM ointment at bedtime throughout the treatment period).

Images were gathered from subject #2 early in treatment (four weeksafter the initiation of treatment) and at a later time point duringtreatment (twelve weeks after the initiation of treatment) (autologousserum eight times a day and loteprednol four times a day for four weeks,then loteprednol twice a day for two weeks, then loteprednol once a dayfor the long-term).

Images were gathered from subject #3 at a time point early in treatment(eight weeks after the initiation of treatment) and at a later timepoint (nine months after the initiation of treatment) (artificialtears).

Laser scanning in vivo confocal microscopy (Heidelberg Retina Tomograph3 with the Rostock Cornea Module, Heidelberg Engineering GmbH,Dossenheim, Germany) of the central cornea was performed in thesesubjects. The microscope used a 670-nm red wavelength diode laser sourceand was equipped with a 63× objective immersion lens with a numericalaperture of 0.9 (Olympus, Tokyo, Japan). The laser confocal microscopeprovided images that represent a coronal section of the cornea of400×400 μm, which is 160,000 μm² at a selected corneal depth, andseparated from adjacent images by approximately 1 to 4 μm with a lateralresolution of 1 μm/pixel. Digital images were stored on a computerworkstation at 30 frames per second. A disposable sterilepolymethylmethacrylate cap (Tomo-Cap; Heidelberg, Engineering GmbH,Dossenheim, Germany) filled with a layer of hydroxypropylmethylcellulose 2.5% (GenTeal gel; Novartis Ophthalmics, East Hanover,N.J.) in the bottom, was mounted in front of the cornea module opticsfor each examination. One drop of topical anesthesia 0.5% proparacainehydrochloride (Alcaine; Alcon, Fort Worth, Tex.) was instilled in botheyes, followed by a drop of hydroxypropyl methylcellulose 2.5% (GenTealgel, Novartis Ophthalmics) in both eyes. One drop of hydroxypropylmethylcellulose 2.5% was also placed on the outside tip of the cap toimprove optical coupling. The tip of the cap was manually advancedtowards each patient's eye until the gel contacted the central surfaceof the cornea.

A total of six to eight volume and sequence scans were obtained from thecenter of each cornea, at least three of which were sequence scans withparticular focus on the subepithelial area, the subbasal nerve plexus,and epithelial dendritic cells, typically at a depth of 14 to 81 μm. Arepresentative image(s) were selected for analysis. The images wereselected from the layer immediately at or posterior to the basalepithelial layer and anterior to the Bowman's layer. The criteria toselect the images were the best focused and complete images, with thewhole image in the same layer, without motion, without folds, and goodcontrast.

Epithelium reflectivity was assessed by the area of hyperreflectivecells compared to the area of normal cells, and quantitating the amountof hyperreflective nuclei of cells in a single frame (400 μm×400 μm).Epithelial cell size and density was analyzed by manual measurement ofthe area of the cell and the number of cells in a frame. Both parameterswere measured using ImageJ software and the software provided with themicroscope. Hyperreflective cells were measured and expressed as apercentage. As is known in the art, the epithelial layer depth may haveminor variation from subject to subject or within the area of the corneain a single subject. The morphology of cells present in the epitheliallayer can be used to identify with precision the layer of the cornea tobe analyzed.

The in vivo confocal images gathered from each subject were examined.The images demonstrate that, in patients that show a good response totreatment for his or her dry eye syndrome, there is a decrease in thenumber or percentage of hyperreflective superficial epithelial cellspresent in the center of the cornea, a decrease in the average size ofsuperficial epithelial cells present in the center of the cornea, and anelevation in the density of superficial epithelial cells present in thecenter of the cornea following treatment or at a later time point intreatment as compared to an earlier time point (prior to treatment or atan earlier time point in treatment) in the same subject (compare FIG. 2to FIG. 1 (human subject #1); FIG. 4 to FIG. 3 (human subject #2), FIG.6 to FIG. 5 (human subject #3).

These data indicate that efficacy of treatment of dry eye syndrome inhumans can be determined by detecting one or more of the change in thenumber or percentage of hyperreflective superficial epithelial cellspresent in the cornea (e.g., in the center of the cornea), the change inthe average size of superficial epithelial cells present in the cornea(e.g., in the center of the cornea), and the change in the density ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea), where one or more of a decrease in the number orpercentage of hyperreflective superficial epithelial cells present inthe cornea (e.g., in the center of the cornea), a decrease in theaverage size of superficial epithelial cells present in the cornea(e.g., in the center of the cornea), and an elevation in the density ofsuperficial epithelial cells present in the cornea (e.g., in the centerof the cornea) following treatment or at a later time point duringtreatment as compared to an earlier time point (a time point prior totreatment or at an earlier time point during treatment) in the samesubject indicate that the treatment is effective.

Example 2. Detection of Changes in the Dendritic Cells Present in theCentral Cornea of Patients Having Dry Eye Syndrome Upon Treatment

In vivo confocal microscopy was performed to image the dendritic cellspresent in the center of the corneas of two subjects (human subjects #1and #2; described above) having dry eye syndrome (i) before or at anearly time point in therapy, and (ii) at a later time point in therapy.Each of these patients demonstrated a good response to treatment for hisor her dry eye syndrome.

Laser scanning in vivo confocal microscopy in these subjects wasperformed as described in Example 1. The resulting confocal images fromtwo subjects were evaluated for density of immune cells present in thecenter of the cornea, the average size of dendritic immune cells presentin the center of the cornea, and the average area covered by dendriticimmune cells present in the center of the cornea. In vivo confocalmicroscopy images at a depth of 45 to 72 μm at the level of basalepithelial layers, basal lamina, or subbasal nerve plexus were chosenfor analysis of dendritic cells. It is noted that the exact identity ofthe dendritic cells cannot be specified, as they could be monocytes ortissue macrophages, but most likely dendritic cells. Dendritic cellswere morphologically identified as bright individual dendriformstructures with cell bodies. The number of dendritic cells can becounted using software (Cell Count, Heidelberg Engineering GmbH) in themanual mode. The dendritic cell size and number of dendrites perdendritic cell can be analyzed using ImageJ software (available at NIHwebsite). Dendritic cell size can be measured as the area covered by asingle cell.

The in vivo confocal images gathered from each subject were examined.The images demonstrate that, in patients that show a good response totreatment for his or her dry eye syndrome, there is a decrease in thedensity of dendritic immune cells present in the center of the cornea, adecrease in the average size of dendritic immune cells present in thecenter of the cornea, and a decrease in the average area covered bydendritic immune cells present in the center of the cornea followingtreatment as compared to prior to treatment or at an earlier time pointin the treatment of the same subject (compare FIG. 8 to FIG. 7 (humansubject #1); and FIG. 10 to FIG. 9 (human subject #2)).

These data indicate that efficacy of treatment of dry eye syndrome inhumans can be determined by detecting one or more of the change indensity of dendritic immune cells present in the cornea (e.g., in thecenter of the cornea), the change in the average size of dendriticimmune cells present in the cornea (e.g., in the center of the cornea),and the change in the average area covered by dendritic immune cellspresent in the cornea (e.g., in the center of the cornea), where one ormore of a decrease in the density of dendritic immune cells present inthe cornea (e.g., in the center of the cornea), a decrease in theaverage size of dendritic immune cells present in the cornea (e.g., inthe center of the cornea), and a decrease in the average area covered bydendritic immune cells present in the cornea (e.g., in the center of thecornea) following treatment or at a later time point during treatment ascompared to an earlier time point (e.g., a time point prior to treatmentor at an earlier time point during treatment) in the same subjectindicate that the treatment is effective.

Example 3. Detection of Changes in the Corneal Nerves in Patients HavingDry Eye Syndrome Upon Treatment

In vivo confocal microscopy was performed to image the nerve cellspresent in the corneas of two subjects (human subjects #1 and #2;described above) having dry eye syndrome (i) before or at an early timepoint in therapy, and (ii) at a later time point in therapy. Each ofthese patients demonstrated a good response to treatment for his or herdry eye syndrome.

Laser scanning in vivo confocal microscopy in these subjects wasperformed as described in Example 1. The nerve analysis can be doneusing the semi-automated tracing program NeuronJ (Meijering et al.,Cytometry A 58:167-176, 2004), a plug-in for ImageJ (available at theimagescience.org website). Nerve density can be assessed by measuringthe total length of the nerve fibers in micrometers per frame (160,000μm²). Main nerve trunks are defined as the total number of main nervetrunks in one image after analyzing the images anterior and posterior tothe analyzed image to confirm that these did not branch from othernerves. Nerve branching is defined as the total number of nerve branchesin one image. The number of total nerves measured is defined as thenumber of all nerves, including main nerve trunks and branches in oneimage. The grade of nerve tortuosity can be classified in four gradesaccording to a tortuosity grading scale reported by Oliveira-Soto andEfron (Cornea 20:374-384, 2001).

The in vivo confocal images gathered from each subject were examined.The images demonstrate that, in patients that show a good response totreatment for his or her dry eye syndrome, there is an elevation in thedensity or average length of nerves present in the cornea, an elevationin the amount of branching in nerves present in the cornea, and anelevation in the total number of nerves present in the cornea followingtreatment as compared to prior to treatment or at an earlier point oftreatment in the same subject (compare FIG. 12 to FIG. 11 (human subject#1); and FIG. 14 to FIG. 13 (human subject #2)).

These data indicate that efficacy of treatment of dry eye syndrome inhumans can be determined by detecting one or more of the change in thedensity or average length of nerves present in the cornea, the change inthe amount of branching in nerves present in the cornea, and the changein the total number of nerves present in the cornea, where one or moreof an elevation in the density or average length of nerves present inthe cornea, an elevation in the amount of branching in nerves present inthe cornea, and an elevation in the total number of nerves present inthe cornea following treatment or at a later time point during treatmentas compared to an earlier time point (e.g., a time point prior totreatment or at an earlier time point during treatment) in the samesubject indicate that the treatment is effective.

Based on the data in this and the above examples, a physician canselectively prescribe, administer, or recommend a therapeutic treatmentto a subject having dry eye syndrome based on ocular physical parametersdetermined using in vivo confocal microscopy (see, for example, thetreatment flow chart shown in FIG. 15).

Example 4. Detection of Changes in the Eyes of Patients Having Acute orChronic Allergy

In vivo confocal microscopic images were gathered from the eyes ofnormal patients and patients having acute or chronic allergy. Thespecific eye structures analyzed were superficial epithelial cells inthe central cornea, epithelial cells in the conjunctiva, dendritic cellsin the conjunctiva, blood vessels in the conjunctiva, lymph vessels inthe conjunctiva, dendritic cells in the peripheral cornea, and dendriticcells in the central cornea.

In vivo confocal microscopic analysis of superficial epithelial cells inthe central cornea were imaged as described above in Example 1. Theresulting data show that subjects having either acute or chronic allergyhave an increase in hyperreflective epithelial cells in the centralcornea (e.g., an increase in the tight junction and squamous metaplasialight reflectivity) and an increase in the average epithelial cell sizein the central cornea as compared to a normal (healthy) subject (FIG.16; compare the center and right panels to the left panel).

In vivo confocal microscopic analysis of the conjunctival epithelium wasgenerally performed as described in Example 1, except that themicroscope was aimed at the bulbar and the tarsal conjunctiva. Theimages were recorded, and the three best focused images were selectedfor analysis. The resulting data show that a subject having acuteallergy has an elevation in the reflectivity of epithelial cells in theconjunctiva (e.g., elevated reflectivity of the tight conjunctions) ascompared to a normal (healthy) control (FIG. 17; compare the centerpanel to the left panel). Although epithelial cells in the conjunctivaare naturally reflective, the data show a distinct elevation in thelevel of reflectivity of these epithelial cells in a subject havingacute allergy.

In vivo confocal microscopic analysis of conjuctival dendritic cells wasgenerally performed as described in Example 1, except that themicroscope was aimed at the subepithelial layers of the bulbar and thetarsal conjunctiva. The images were recorded and the three best focusedimages were selected to determine dendritic immune cell number andmorphology. The resulting data show that subjects having acute orchronic allergy have an elevation in the density of dendritic cells inthe conjunctiva, an elevation in the average dendritic cell size in theconjunctiva, and an elevation in the number or percentage ofhyperreflective dendritic cells in the conjunctiva as compared to ahealthy control (FIG. 18; compare the center and right panels to theleft panel).

In vivo confocal microscopy of conjunctival blood vessels was generallyperformed as described in Example 1, except that the microscope wasaimed at the blood vessels of the bulbar conjunctiva. The properties ofthe blood vessels, and the number and behavior (e.g., sticking to theblood vessel wall) of inflammatory cells, identified as round-shapedhyperreflective corpuscles was analyzed. The resulting data show that asubject having acute allergy has increased dilation in the lumen ofblood vessels in the conjunctiva, an elevation in the average size ofinflammatory cells present in the conjunctiva, and increased sticking(e.g., increased time of transient residence) of inflammatory cells tothe blood vessel wall in the conjunctiva as compared to a normal(healthy) control (FIG. 19; compare the right panel to the left panel).

Conjunctival lymphatic vessels were also imaged using in vivo confocalmicroscopy. This imaging was also performed as generally described inExample 1, except that the microscope was aimed at the lymphatic vesselsof the bulbar conjunctiva. The resulting data show that a subject havingacute allergy has an elevated number of inflammatory cells present inlymphatic vessels in the conjunctiva and an elevation in the averagesize of the inflammatory cells present in lymphatic vessels in theconjunctiva as compared to a normal (healthy) subject (FIG. 20; comparethe right panel to the left panel).

Dendritic cells in the peripheral cornea were also imaged using in vivoconfocal microscopy (performed as described in Examples 1 and 2). Thedata gathered show that a subject having acute allergy has an elevationin dendritic cell density in the peripheral cornea, an elevation in theaverage dendritic cell size in the peripheral cornea, and an elevationin the number of hyperreflective dendritic cells as compared to a normal(healthy) subject (FIG. 21; compare the right panel to the left panel).

Dendritic cells in the central cornea were also imaged using in vivoconfocal microscopy (performed as described in Examples 1 and 2). Theresulting data show that a subject having acute allergy has an elevationin the average dendritic cell size in the central cornea as compared toa normal (healthy) subject (FIG. 22; compare the right panel to the leftpanel).

These data indicate that in vivo confocal microscopy can be used toidentify subjects that can benefit from a specific therapeutictreatment. For example, in vivo confocal microscopic imaging can beperformed on a subject, and the subject administered, prescribed, orrecommended a specific treatment based on the physical substructures inthe eye detected using in vivo confocal microscopy (e.g., any of thesubstructures described herein).

Example 5. Study of Ocular Physical Parameters in Subjects with Dry EyeSyndrome

Additional experiments were performed to identify additional ocularstructural changes that appear in subjects having dry eye syndrome.

Materials and Methods

Twenty-four eyes of 24 patients with dry eye syndrome (DES) and 15 eyesof 15 normal volunteers were included in the study. A detailed clinicalhistory and slit lamp biomicroscopy examination was performed for eachparticipant. DES was diagnosed based on symptoms (foreign body sensationand dryness of the eye) and a Schirmer I test result of <10 mm/5minutes. Tear break-up time (TBUT) was recorded as the average of threesuccessive measurements. The corneal fluorescein staining score wasevaluated based on grades 0 to 3 in each of the four quadrants for atotal score of 12, according to the National Eye Institute gradingscale. All normal control subjects did not have any ocular irritation,did not use any ocular medication, and had normal Schirmer test (>10mm/5 minutes) and normal slit lamp biomicroscopy exam. The exclusioncriteria included history of ocular trauma, ocular surgery, contact lensuse, drug allergy, diabetes, or the presence of other systemic or oculardisease except dry eye.

In vivo slit scanning confocal microscopy (Confoscan 4; NidekTechnologies, Gamagori, Japan) was performed in the central cornea ofall subjects as has described herein. The contralateral eye was fixed toa light source to stabilize the patient's view. The microscope wasequipped with a 40×/0.75 objective lens. One drop of topical anesthesiaof 0.5% proparacaine hydrochloride (Alcaine, Alcon) was instilled inboth eyes. A drop of 0.3% hypromellose (GenTeal gel, Novartis) wasapplied as coupling medium between the tip of the objective lens and thecornea. Full thickness confocal scans were acquired at a speed of 25frames per second, obtaining 350 images per scan, every 7 μm. A secondscan was obtained for the anterior cornea, obtaining sections every 3μm. Each image represented a coronal section of 460×345 μm with aminimum axial step of 1 μm, magnification of 500×, and lateralresolution of 1 μm/pixel. A total of 4 to 8 scans were obtained for eachcornea by the same experienced operator in all subjects, depending onfull thickness or anterior scan mode.

A minimum of 3 representative images of the superficial epithelium, ofthe basal epithelium, and of the subbasal nerve plexus were selected foranalysis for each eye. Two masked observers evaluated the in vivoconfocal images for superficial epithelial cell density, superficialepithelial cell size, number of hyperreflective cells in the superficialepithelial cell layer, basal epithelial cell density, and analyzed thecorneal subbasal nerve plexus. Briefly, nerve density was assessed bymeasuring the total length of the nerve fibers in micrometers per mm²(area of interest=0.1335 mm²). Main nerve trunks were defined as thetotal number of main nerves in one image. Nerve branching was defined asthe total number of nerve branches in one image. The number of totalnerves measured was defined as the number of all nerves, including mainnerve trunks and branches in one image. The grade of nerve tortuositywas classified in four grades according to a tortuosity grading scalereported by Oliveira-Soto et al. (Cornea 20:374-384, 2001). Statisticalanalysis was performed with SAS software version 9.2 (SAS InstituteInc., Cary, N.C., USA). Data from one randomly chosen eye per person wasused for analysis. Data distribution and homogeneity of variance wereanalyzed. Comparisons were performed by two-tailed t-test andcorrelations were assessed by Pearson correlation coefficient.Differences were considered statistically significant for p-values lessthan 0.05.

Results

Twenty-four eyes of 24 patients with diagnosis of DES were included foranalysis in the study and were compared with 15 eyes of 15 normalvolunteers. The mean age of the DES patients was 55.1±19.1 with amale/female ratio of 10/14, and in the control group the mean age was59±17 with a male/female ratio of 8/7. The clinical evaluation of DESwas apparent in the DES group compared to the normal controls: TBUT(3.0±1.9 vs. 11.7±1.0; p<0.0001), Schirmer I test (3.3±2.2 vs. 13.1±1.0;p<0.0001), and corneal staining (3.2±1.4 vs. 0±0; p<0.0001). Thedemographics and clinical data of the DES group and control group areshown in Table 1.

TABLE 1 Demographic data of normal controls and patients with dry eyesyndrome. Controls Dry Eye Syndrome Number of patients (n) 15 24 Age(mean ± SD) (years)   59 ± 17 55.1 ± 19.1 Gender (male/female) 8/7 10/14Tear break up time (TBUT) (seconds) 11.7 ± 1.0 3.0 ± 1.9 Schirmer I test(mm) 13.1 ± 1.0 3.6 ± 2.0 Corneal fluorescein staining score  0 3.3 ±1.5 Values are mean ± standard deviation (SD).

The mean nerve parameters and corneal epithelial cell parameters for DESand normal control groups are shown in Table 2.

In the group of patients with DES, the corneal subbasal nerve plexus wassignificantly reduced (FIG. 23). Specifically, the mean total nervelength (5,036.0±2,459.2 vs. 15,690.6±6,695.1 μm/mm2), total number ofnerves (5.1±1.9 vs. 12.5±3.5; p<0.0001), main nerve trunks (2.9±1.1 vs.4.6±1.1; p<0.0001), and number of branches (1.8±1.0 vs. 7.8±3.4;p<0.0001) were significantly lower as compared to controls (FIGS.24-27). The nerve tortuosity was slightly increased in DES compared tocontrols, but did not reach statistical significance (1.9±0.7 vs.1.6±0.3; p=0.124) (data not shown).

Patients with DES had a significant decrease in superficial epithelialcell density (1645.4±302.8 vs. 1993.5±448.9; p=0.003) and an increase intheir size (660.8±123.2 vs. 410.7±53.0; p<0.0001) with a higher numberof hyperreflective cells (229.0±70.0 vs. 115.6±93.1; p<0.0001) ascompared to controls. The basal epithelial cell density wassignificantly increased in DES patients as compared to controls(6248.5±741.6 vs. 5718.3±367.8; p=0.002) (FIGS. 28-30).

TABLE 2 Corneal epithelial cell parameters and corneal subbasal nerveplexus parameters in control and dry eye syndrome groups. Control DryEye p-value Epithelial cell parameters Superficial epithelial density(cells/mm²) 1993.5 ± 448.9 1645.4 ± 302.8  *p = 0.003 Superficialepithelial cell size (μm²) 410.7 ± 53.0 660.8 ± 123.2 *p < 0.001Hyperreflectivity cells (cells/mm²) 115.6 ± 93.1 229.0 ± 70.0  *p <0.001 Basal epithelium (cells/mm²) 5718.3 ± 367.8 6248.5 ± 741.6  *p =0.002 Corneal subbasal nerve plexus parameters Total Nerve length(μm/mm²) 15,690.6 ± 6,695.1 5,035.0 ± 2,459.2 *p < 0.001 (μm/frame)2,094.7 ± 893.8  672.3 ± 328.3 Total number of Nerves (n^(o)/frame) 12.5± 3.5 5.1 ± 1.9 *p < 0.001 Main Nerves (n^(o)/frame)  4.6 ± 1.1 2.9 ±1.1 *p < 0.001 Branching (n^(o)/frame)  7.8 ± 3.4 1.8 ± 1.0 *p < 0.001Tortuosity  1.6 ± 0.3 1.9 ± 0.7 p = 0.124 Values reported as mean ±standard deviation. *Statistically significant (p < 0.05) compared tocontrols.

Pearson correlation was performed to determine significant correlationsbetween the corneal nerves and the epithelial parameters, and to assessassociations between the in vivo confocal microscopy findings and theclinical tests performed to evaluate DES, such as corneal fluoresceinstaining score, TBUT, and Schirmer's test.

There was an inverse correlation between the cell size of thesuperficial corneal epithelium and some of the corneal nerve parameters(FIGS. 31-34 and 36), and a positive correlation between the number ofnerve branches and the superficial epithelial cell density (FIG. 35).Superficial epithelial cell size was significantly correlated to totalnerve length (R=−0.71, p<0.0001), total number of nerves (R=−0.68,p<0.0001), main nerve trunks (R=−0.57, p<0.0001) and nerve branches(R=−0.74, p<0.0001). The density of the basal epithelial was alsoinversely correlated to total nerve length (R=−0.38, p=0.008) (FIG. 36).

In patients with DES, increased corneal fluorescein staining scorecorrelated with an increase in superficial epithelial cell size (R=0.54,p=0.007) (FIG. 37), while increased corneal fluorescein staining scoreshowed an inverse correlation with basal epithelial cell density (FIG.38) and superficial epithelial cell density (FIG. 39). A correlation wasnot observed between TBUT and Schirmer's test with the in vivo confocalmicroscopy findings. These data indicate a number of other opticalphysical parameters that can also be used to diagnose a subject ashaving dry eye syndrome.

The TBUT, corneal fluorescein staining score, and Ocular Surface DiseaseIndex (OSDI) Questionnaire Score was determined for the subjects havingdry eye syndrome before anti-inflammatory treatment (Pre-Rx), at a firstfollow-up visit following initiation of anti-inflammatory treatment(2.3±0.9 months after initiation of treatment), and at a secondfollow-up visit following initiation of anti-inflammatory treatment(4.8±1.8 months following initiation of anti-inflammatory treatment)(FIG. 40). Pearson correlation was performed to assess the correlationsbetween the immune cell parameters and the acinar density and theclinical tests performed to evaluate dry eye syndrome, such as TBUT andcorneal fluorescein staining. The resulting data show a negativecorrelation between corneal epithelial immune cell density and TBUT, anegative correlation between intraglandular immune cell concentration (%lumen occupied) and TBUT, and a negative correlation between acinardensity and corneal fluorescein staining (FIG. 41). These data furtherindicate that corneal epithelial immune cell density, intraglandularimmune cell concentration, and acinar density can be used to assess theefficacy of treatment of dry eye disease in a subject.

Example 6. Immune Cell Alternations in the Cornea and the Effect ofAnti-Inflammatory Treatment on Different Subtypes of Dry Eye Syndrome

These experiments were performed to determine if the alternations incorneal immune cells occur in different subtypes of dry eye syndrome,and if subjects having these different subtypes of dry eye syndrome areresponsive to anti-inflammatory treatment.

Materials and Methods

This was a retrospective, case-control study of sixty-six eyes ofpatients having dry eye syndrome, and 28 normal eyes. The exclusioncriteria for both the case and control groups included diabetesmellitus, refractive surgery, and corneal infection. Based on the dryeye subtype, the patients were divided into three groups:aqueous-deficient, evaporative, and mixed mechanism. Laser in vivoconfocal microscopy (as described herein) using a HRT 3 and CorneaRostock Module was performed on the central cornea of patients with dryeye syndrome and controls (using the methods described herein). Threerepresentative images (n=3) from each eye were analyzed in a blindedfashion for: dendritic cell density defined as hyperreflectivedendriform cells, dendritic cell size defined as the average area ofeach dendritiform cell, surface of the cornea covered by dendriticcells, and the number of dendritic processes per cell. Exemplary in vivoconfocal microscopic images showing the central corneal of an eye from anormal subject, an eye from a subject having evaporative dry eyesyndrome, an eye from a subject having mixed mechanism dry eye syndrome,and an eye from a subject having aqueous-deficient dry eye syndrome areshown in FIGS. 42A-D.

The resulting data show that patients with aqueous-deficient dry eyesyndrome (density: p=0.001; cell area: p=0.0007; covered area; p=0.009;number of dendrites: p=0.0001) and mixed dry eye syndrome patients(density: p=0.03; cell area: p=0.01; covered area: p=0.04; number ofdendrites: p=0.0003), but not evaporative dry eye syndrome patients(density: p=0.48; cell area: 0.31; area covered: p=0.45; number ofdendrites: p=0.50), had significant increases for all four parameters ascompared to controls (FIGS. 43-46).

The cornea immune cell density (cells/mm²) and area (μm²) was determinedin the eyes of the subjects having dry eye syndrome receiving or notreceiving anti-inflammatory treatment. The data show that dry eyesyndrome patients on anti-inflammatory therapy (AIT) had a 20% lowerdendritic cell density and a 15% decrease in the dendritic cell size andarea of the cornea covered by dendritic cells compared to dry eyesyndrome patients not receiving anti-inflammatory therapy (AIT) (FIG.47). In sum, these data indicate that in vivo confocal microscopyenables the detection of significant dry eye syndrome-induced changes inimmune cells in the cornea in subjects having different subtypes of dryeye syndrome, and show that dendritic cell density and morphology canserve as a parameter to assess the severity of dry eye syndrome and theefficacy of treatment of dry eye syndrome.

Example 7. Use of Confocal Microscopy to Determine Efficacy ofAutologous Serum Treatment in Subjects Having Dry Eye Syndrome

This set of experiments was performed to determine whether in vivoconfocal microscopy can be used to determine the efficacy of treatmentof dry eye syndrome in subjects following treatment with autologousserum. Specifically, the study was designed to evaluate the effect ofautologous serum eye drops on the corneal subbasal nerve plexus for thetreatment of severe dry eye syndrome.

Central corneal images were collected using in vivo confocal microscopyas described herein (using an scanning laser in vivo confocal microscopewith the Heidelberg Retina Tomograph 3 with the Rostock Cornea Module(Heidelberg Engineering GmbH) in each subject at baseline and at eachfollow-up visit. The autologous serum was prepared by obtaining a venousperipheral blood sample from the subject to be treated, and centrifugingthe sample for 10 minutes at 3000 RPM. The resulting supernatant serumwas removed and diluted to a concentration of 20% serum in balancedsaline solution. Five-mL aliquots of the autologous serum solution wereplaced into sterile dropper bottles and stored until use at −2° C. Eachbottle was used for 1 week, with 1 drop administered to each eye 6 to 8times a day, and stored during weekly use at 6° C.

One eye from each treated subject was randomly selected and threerepresentative images of the central corneal nerve plexus were analyzedstarting from baseline to the last follow-up visit. In each obtainedimage, the number of nerves, the length of the nerves, number of mainnerve trunks, and the number of nerve branches were determined. Theseparameters were analyzed by two masked observers using the Image Jsoftware (NIH). The results were subjected to statistical analysis usingthe Stata Software package 11.2 (T-test, multivariate and logisticregression analysis). A total of 57 patients with severe dry eyesyndrome, and unresponsive to maximal conventional therapy were includedin this study, and were compared to a group of normal controls. Thedemographics of the dry eye syndrome patients and the healthy (normal)control group are shown in Tables 3 and 4 below.

TABLE 4 Demographics of the Healthy (Normal) Control Group Number 25 Age(years) 33.1 ± 7.6  Range (years) 22-60 Sex (M:F)% 10 (50%):10 (50%)Prior to treatment, the corneal subbasal nerve plexus (as assessed bydetermining the total nerve length) was significantly decreased in dryeye syndrome patients as compared to normal controls (FIG. 48; *,p<0.001). An increase in the number of corneal nerves was detected asearly as 3 months after the initiation of treatment with autologousserum as compared to the number of corneal nerves detected in the samepatients prior to treatment with autologous serum (FIG. 49). It was alsoobserved that the nerve plexus varied among the dry eye patients atbaseline (prior to treatment with autologous serum) (FIG. 50). After

TABLE 5 Demographics of Dry Eye Syndrome Patients using Autologous SerumDrops Age (years) 57.7 ± 2.3  Range (years) 22-87 Sex (M:F)% 11 (19%):46(81%) Evaporative DES  8 (14%) Aqueous Deficient DES 5 (9%) MixedMechanism DES 26 (45%) Neurotrophic keratopathy 5 (9%) GVHD 13 (22%)Follow up period (months) 8.0 ± 0.7 Range (months)  2.1-21.7treatment with autologous serum, there was a significant increase in thenerve plexus in patients having dry eye syndrome (FIG. 51). The totalnerve length and total nerve number in the corneas of subjects havingdry eye syndrome were increased after the use of autologous serum ascompared to the baseline values (prior to autologous serum treatment) inthese subjects (FIGS. 52 and 53;

, p<0.001). The main nerve trunk length and the main nerve trunk numberalso increased significantly in the corneas of subjects having dry eyesyndrome after treatment with the autologous serum as compared to thebaseline values (prior to autologous serum treatment) in these subjects(FIGS. 54 and 55;

, p<0.001). The nerve branch length and number also increasedsignificantly in the corneas of subjects having dry eye syndrome aftertreatment with the autologous serum as compared to the baseline values(prior to autologous serum treatment) in these subjects (FIGS. 56 and57;

, p<0.001; *, p<0.001). The data further show that 90% of the patientshaving dry eye syndrome demonstrated nerve regrowth following treatmentwith autologous serum (FIG. 58). Overall, the patients having dry eyesyndrome that were treated with autologous serum had a mean increase of65% of the cornea nerve plexus (FIG. 59). It was also observed that agehad a significant negative correlation with the corneal nerve regrowthin patients having dry eye syndrome after treatment with autologousserum (FIG. 60). Nerve regeneration was observed in patients having dryeye syndrome following treatment with autologous serum, regardless ofthe underlying cause of dry eye syndrome (FIG. 61).

The patients having dry eye syndrome that were treated with autologousserum showed a significantly improvement in TBUT as compared to thebaseline values (prior to autologous serum treatment) in these subjects(FIG. 62; *, p<0.003). Overall, patients having dry eye syndrome thatwere treated with autologous serum had a mean increase of 64% in TBUT(FIG. 63). In addition, patients having dry eye syndrome that weretreated with autologous serum showed a significant decrease in OSDIQuestionnaire Score as compared to the baseline values (prior toautologous serum treatment) in these subjects (FIG. 64). Overall, 79% ofthe patients having dry eye syndrome that were treated with autologousserum had a decrease in their OSDI score, with a mean overall decreasein OSDI score of 12% (FIG. 65).

In sum, these data show that autologous serum is an effective treatmentas coadjuvant therapy in severe dry eye syndrome. In addition, thesedata demonstrate that changes in the corneal epithelium (as detectedusing in vivo confocal microscopy) can be detected as used to assess theefficacy of treatment of dry eye syndrome in a subject.

Example 8. Cellular Changes in the Cornea and Conjunctiva in Allergy andNon-Allergic Ocular Inflammatory Diseases

These experiments were performed to determine whether changes in thecornea and conjunctiva occur in ocular allergy and non-allergic ocularinflammatory diseases.

In these experiments, the conjunctiva, and central and peripheral corneaof six patients (8 eyes) with ocular allergy (OA), 9 patients (10 eyes)with non-allergic ocular inflammatory diseases (OID), and six control(healthy) subjects (8 eyes) were imaged using scanning laser in vivoconfocal microscopy (HRT3/RCM set-up and performed as described herein)with 2 sequences per area. The images were quantified for dendritiformcell (DC) density, superficial epithelial (SE) cell reflectivity, andsuperficial epithelial cell border reflectivity. Conjunctival bloodvessels were assessed for the presence and adhesion of immune cells tovessel wall (cells/100 μm).

The mean age of the subjects having ocular allergy was 25.5±7.7 years,the mean age of the subjects having an ocular inflammatory disease was54±10.5 years, and the mean age of the control subjects was 30.6±6.4years. The OID group consisted of patients having infectious keratitis,foreign body infiltration, chlamydial conjunctivitis, graft versus hostdisease, Steven Johnson's syndrome, and marginal keratitis. All allergypatients in these experiments suffered from seasonal or perennialallergies, with 2 out of the 8 eyes presenting with acute allergicconjunctivitis.

Table 6 shows the cornea superficial epithelial cell reflectivity andcorneal dendriform cell density in ocular allergy, non-allergic ocularinflammatory, and control groups. As can be seen in the table below,patients with OA and OID demonstrated increased superficial epithelialcell hyperreflectivity in the cornea (71%±6.9 and 78%±4.3, respectively;mean±SEM), as compared to controls (0% and 3.5%±1,3; p<0.0001). Thesuperficial epithelial cell border reflectivity in the area of tightjunctions was increased in the cornea for OA and OID (83%±5.0 and84%±4.5; mean±SEM) and conjunctiva (77%±5.8 and 70%±5.7), as compared tocontrols (0% cornea; 11%±4.0 conjunctiva; p<0.0001). Central cornealdendritic cell density was increased for OA subjects (3.5×; 67.5±13.7;fold increase; mean±SEM) and for OID subjects (6.4×; 125±46), ascompared to control subjects (19.5±3; p=0.11). While central cornealdendritic cell density was lower in subjects with acute OA (46.8±21.9cells/mm²; mean±SEM) versus subjects with acute OID (256.25±175),subjects with chronic OA (75.0±16.7) had higher dendritic cell densitycompared to subjects with chronic IOD and normal subjects (p=0.04). Inthe peripheral cornea, a 35% and 90% increase in dendritic cell densitywas observed in subjects with acute OA and chronic OA, respectively, ascompared to controls (p>0.05). Data showing the mean percentage ofreflective superficial epithelial cells in the cornea and conjunctivafor controls subjects, subjects having OA, and subjects having OID areshown in FIG. 66. Data showing the mean percentage of superficialepithelial cells in the cornea and conjunctiva showing borderreflectivity for control subjects, subjects having OA, and subjectshaving OID are shown in FIG. 67. Data showing the mean dendritic celldensity in the corneas and conjunctiva of control subjects, subjectshaving OA, and subjects having OID are shown in FIG. 68. Data showingthe mean dendritic cell density in the corneas and conjunctiva ofcontrols subjects, subjects having OA, and subjects having OID are shownin FIG. 69.

Table 7 shows the cojunctival superficial epithelial cell reflectivityand conjunctival dendriform cell density in ocular allergy, non-allergicocular inflammatory, and control groups. Conjunctival dendritic celldensity was significantly increased in both subjects having OA(131.2±100; mean±SEM) and subjects having OID (234.4±206), as comparedto controls (17.5±16.2; p=0.001). Exemplary in vivo confocal microscopicimages of the cornea and the conjunctiva for subjects having OA or OIAare shown in FIG. 70.

TABLE 6 Cornea Superficial Epithelial Cell Reflectivity and CornealDendritiform Cell Density SE SE Border CORNEA Reflectivity ReflectivityCentral DC Peripheral DC Normal 0 0 19.5 ± 3.0  78.7 ± 11.5 ControlsOcular 70.6 ± 6.9* 83.1 ± 5.0*  67.5 ± 13.7 117.2 ± 16.4 Allergy OcularIn- 78.8 ± 4.3* 83.8 ± 4.5* 125.0 ± 46.0 156.2 ± 15.6 flammatory Disease*p < 0.0001 (ANOVA between OA, IOD and controls; Mean ± SEM)

TABLE 7 Conjunctival Superficial Epithelial Cell Reflectivity andConjunctival Dendritiform Cell Density SE SE Border CORNEA ReflectivityReflectivity Central DC Peripheral DC Normal 0 0 19.5 ± 3.0  78.7 ± 11.5Controls Ocular 70.6 ± 6.9* 83.1 ± 5.0*  67.5 ± 13.7 117.2 ± 16.4Allergy Ocular In- 78.8 ± 4.3* 83.8 ± 4.5* 125.0 ± 46.0 156.2 ± 15.6flammatory Disease *ANOVA between OA, OID eyes and controls (*p =0.0001; **p = 0.001; mean ± SEM)Conjunctival vessels demonstrated increased immune cell adhesion insubjects having OA (5.5±0.7 cells/100 μm vessel length; mean±SEM) and insubjects having OID (5.7±1.7), as compared to control subjects(0.13±0.35; p<0.0001) (FIG. 71).

In sum, the data show that profound immune and inflammatory changesoccur in ocular allergic and non-allergic ocular inflammatory diseases,and superficial epithelial cell changes in the corneal and conjunctivalepithelium of patients with both OA and OID. These data indicate that invivo confocal microscopy can be used to stratify patients and assess asubject's responsiveness to treatment.

Example 9. Corneal Analysis of Subjects Having Limbal Stem CellInsufficiency

An additional set of experiments were performed to determine if thecorneal changes described herein also occur in patients having limbalstem cell insufficiency (LSCI). LSCI is a condition in whichinflammation from injury or disease causes significant ocular morbidity.

In these experiments, the clinical charts and images of three patientsdiagnosed with LSCI were retrospectively reviewed. Each subjectunderwent a complete exam and in vivo confocal microscopy (usingHRT3/RCM, as described herein) of the central cornea and the superiorlimbus at each visit. The patients were followed monthly untilresolution. The patients were treated with topical steroid drops basedon in vivo confocal microscopy findings that indicate the presence ofinflammation. Three representative images from the central cornea andthe superior limbus were selected for the analysis of each parameter,and compared with images obtained from previously tested normal(healthy) subjects. The images were quantified for dendritiform immunecell density (DC) and total counts of nerves and branches withrespective density with ImageJ/Neuron J software.

The first patient (Patient 1) was a 13 year old female who had beenwearing contact lenses for 2 years. Patient 1 had a visual acuity of20/25 OD and 20/40 OS at presentation. After treatment with rimexolone1% QID initially, and then taper for 5 months, Patient 1's visual acuityreturned to 20/20 OU.

The second patient (Patient 2) was a 31 year old female who had beenwearing contact lenses for 15 years. Patient 2 had a visual acuity of20/20 OD and 20/60 OS at presentation. After treatment with loteprednol0.5% QID initially, and then taper for 11 months, Patient 2's visualacuity returned to 20/20 OU.

The third patient (Patient 3) was a 39 year old female who had beenwearing contact lenses for 15 years. Patient 3 had a visual acuity of20/30 OD and 20/20 OS at presentation. After treatment with loteprednol0.5% QID initially, and then taper for 4 months, Patient 3's visualacuity returned to 20/20 OU.

In the examined LSCI subjects, the average length of contact lens wearwas 10.7 years. The subjects were followed for 4-11 months (average of6.7 months). The average visual acuity for the subjects was 20/30 (rangeof 20/20 to 20/60) at presentation.

In vivo confocal microscopy demonstrated that the LSCI subjects had amean central cornea dendritic cell density of 577.4±323.8 cells/mm² atpresentation, which was significantly higher than normal subjects(42.5±40.5, p<0.05) (FIG. 72). In view of the increased presence ofdendritic cells in the central cornea (increased dendritic cell densityin the cornea as compared to normal healthy controls), these subjectswere identified as having corneal inflammation and were administeredloteprednol etabonate (0.5% taper; 2 patients; 4 eyes) or rimexolone (1%taper; 1 patient; 2 eyes) for three months. Following three months oftreatment, the average dendritic cell density in the cornea of the LSCIsubjects significantly decreased to 234.6±233.7 cells/mm² compared tothe dendritic cell density in these subjects on presentation (p=0.001)(FIG. 73). The average density of dendritic cells in the superior limbusafter treatment was 238.2±158.4 cells/mm².

Subjects having LSCI were also determined to have a decrease in cornealnerve count and density at presentation as compared to healthy controls(FIGS. 74 and 75). After treatment, there was a significant increase inthe total number of nerves and branches in the cornea, as well as asignificant increase in nerve branch density as compared to thecorresponding values determined at presentation (FIG. 74).Representative images of a LSCI subject's cornea before and aftertreatment are shown in FIG. 76.

The corneal epithelium in each patient qualitatively showed a higherrate of conjunctivalization by in vivo confocal microscopy, whichsubsequently decreased with treatment (FIG. 77). The in vivo confocalmicroscopic changes observed corresponded to an improvement of meanvisual acuity of 20/20 and a decrease in corneal staining.

These data indicate that LSCI can be diagnosed or the severity of LSCIin a subject can be diagnosed using the in vivo confocal microscopymethods described herein. In addition, the in vivo confocal microscopymethods described herein can be used to determine the efficacy oftreatment of LSCI in a subject.

Example 10. Dendritic Cell Density in Corneas of Healthy Subjects

These experiments were performed to determine the control values fordendritic cell density in the central and peripheral quandrants ofcorneas of healthy control subjects using a laser-scanning in vivoconfocal microscope (Heidelberg Retina Tomograph (HRT3) used inconjunction with the Rostock Cornea Module (RCM), and equipped with 670nm diode laser).

Sixty-five healthy (control) subjects were enrolled in this study.Slit-lamp examination was performed to confirm lack of ocular findingsin the enrolled subjects. Laser in vivo confocal microscopy (usingHRT3/RCM as described herein) was used to gather images of the centerand four quadrants of the cornea. The demographics of the subjectsenrolled in this study are shown in Table 8. Two scan sequences wererecorded for each area.

TABLE 8 Demographics of Normal (Healthy) Subjects in Study Age (years)Normal Subjects mean ± SD Sex (M:F) 65 30.9 ± 10.4 31:34

Exemplary in vivo confocal microscopic images showing the visualizationof the limbus quadrant of the cornea and the peripheral cornea are shownin FIG. 78. Additional exemplary in vivo confocal microscopic images ofeach quadrant of the cornea examined are shown in FIG. 79. A graphshowing the density of dendritic cells in each area of the cornea incontrol healthy subjects is shown in FIG. 80. A graph of the immune celldensity in each area of the cornea in control healthy subjects is shownin FIG. 81. These data reveal the presence of dendritic cells in thecentral and peripheral cornea in all healthy subjects. The dendriticcell density for the central cornea in healthy subjects was 41.01±5.96cells/mm² (mean±SEM). The densities of dendritic cells in healthysubjects in the superior, inferior, and nasal quandrants were higherthan the density of dendritic cells found in the central cornea(mean±SEM; p<0.0001). The superior (96.31±6.10 cells/mm²) and inferior(108.82±9.33) quadrants demonstrate the highest dendritic cell densityas compared to the nasal (60.63±5.15 cells/mm²) and temporal (49.26±3.49cells/mm²) quandrants in the cornea of healthy subjects. There was asignificant statistical difference between the dendritic cell density inthe temporal quadrant and the other peripheral areas in the healthysubjects (p<0.0001). The immune cell density was extremely low overallin the corneas of healthy subjects, with the highest density of immunecells present in the nasal quadrant (1.2±3.8 cells/mm²) and lowestdensity in the central cornea (0.7±2.0 cells/mm²), although nostatistical difference was detected when comparing the five examinedareas of the cornea. Excellent interobserver variability was observedbetween observers for all the areas combined. These values and rangesdetermined in healthy subjects can be non-limiting references values inany of the methods described herein.

Example 11. Additional Study of Dendritic Cells in Corneas of HealthySubjects

An additional study was performed in 85 normal (healthy subjects) (85total eyes) to determine the control values for dendritic cells in thecentral and peripheral quandrants (inferior, nasal, superior, andtemporal peripheral quadrants) of corneas of healthy control subjectsusing a laser-scanning in vivo confocal microscope (Heidelberg RetinaTomograph (HRT3) used in conjunction with the Rostock Cornea Module(RCM), and equipped with 670 nm diode laser) as described herein.Slit-lamp examination was performed to confirm lack of ocular disease ineach subject. The images were assessed for dendritic cell morphology anddensity by two masked observers. Morphology was assessed by number ofdendrites per cell, area of dendritic cells, and dendritic cell field.Statistical analysis was performed with ANOVA/Bonferroni to compare thedifferences between areas and the intraclass coefficient was used toassess interobserver reproducibility.

The mean age of healthy subjects in this study was 31.4 years (range of20 to 69). The images revealed the presence of central (35.6±4.3cells/mm²) and peripheral (74.1±4.5 cells/mm²) corneal dendritic cellsin all areas of all subjects. The number of dendrites, area of dendriticcells, and dendritic cell field in the central cornea were 2.4±0.04dendrites/cell, 71.6±4.3 μm², and 304.2±19.2 μm², respectively. Thenumber of dendrites (3.2±0.07 dendrites/cell, inferior; 2.9±0.03dendrites/cell, nasal; 3.0±0.03 dendrites/cell, superior; and 2.9±0.03dendrites/cell, temporal), area of dendritic cells (134.6±3.5 μm²,inferior; 115.3±3.4 μm², nasal; 117.0±3.5 μm², superior; and 121.2±3.9μm², temporal), and dendritic cell field area (684.4±19.1 μm², inferior;586.2±19.7 μm², nasal; 587.7±17.4 μm², superior; and 601±18.6 μm²,temporal), were found to be significantly larger in all 4 peripheralquadrants (p<0.001). Good interobserver reproducibility coefficient wasfound for dendritic cell area (0.83; 0.77-0.91 95% CI), number ofdendrites per cell (0.97; 0.95-0.99), and dendritic cell coverage area(0.94; 0.85-0.98). These values and ranges determined in healthysubjects can be non-limiting reference values or ranges in any of themethods described herein.

Example 12. Study of Nerves in Corneas of Healthy Subjects

These experiments were performed to determine the subbasal corneal nervedensity in the central cornea and four peripheral quadrants of thecornea of healthy (control) subjects using a laser-scanning in vivoconfocal microscope (Heidelberg Retina Tomograph (HRT3) used inconjunction with the Rostock Cornea Module (RCM), and equipped with 670nm diode laser) as described herein. A total of 37 healthy (control)subjects were used in these studies. Slit-lamp examination was performedto confirm lack of ocular disease in each subject. Three representativeimages were chosen for each corneal area and quantified using NeuronJ, aplugin for ImageJ software (NIH). Two masked observers measured thenumber and density of total nerves, main trunks, and branches for eachimage. Statistical analysis was performed with ANOVA with Bonferronicorrection to compare the differences between the 5 cornea areas. Alinear regression model was applied to assess the changes for gender andage.

The average age of the healthy (normal) subjects participating in thesestudies was 30 years, ranging from 19 to 69 years. The central cornea inthe healthy (control) subjects had a significantly higher number anddensity of total nerves (16.2 nerves/frame, [14.8-17.6] 95% CI; and20067.7 μm/mm², [18776.6-21358.8] 95% CI, respectively), main trunks(3.3, [2.9-3.6] 95% CI), and branches (12.8, [11.4-14.2] 95% CI) incomparison to all peripheral areas (p<0.001). All peripheral areas inthe healthy subjects demonstrated similar distribution of the subbasalnerve plexus for all parameters (p>0.05), including the number anddensity of total nerves in the superior (9.2, [7.8-10.6] 95% CI; and11638.1, [9834.7-13441.5] 95% CI, respectively), inferior (9.2,[7.5-11.0] 95% CI, and 11169.3, [9080.2-13258.4] 95% CI, respectively),temporal (8.9, [7.5-10.3] 95% CI, and 9535.3, [8452.7-10617.9] 95% CI,respectively), and nasal (7.8, [6.2-9.4] 95% CI, and 9621.0,[7832.3-11409.8] 95% CI, respectively) quadrants. An inverse correlationto age (p=0.017), but not for gender (p=0.781) was shown for all nerveparameters. These values and ranges determined in healthy subjects canbe non-limiting references values or ranges in any of the methodsdescribed herein.

Example 13. Additional Study of Nerves in Corneas of Healthy Subjects

Further experiments were performed to determine the subbasal cornealnerve density in the central cornea and four peripheral quadrants of thecornea of healthy (control) subjects using a laser-scanning in vivoconfocal microscope (Heidelberg Retina Tomograph (HRT3) used inconjunction with the Rostock Cornea Module (RCM), and equipped with 670nm diode laser) as described herein. A total of 85 healthy (control)subjects were used in these studies and a total of 85 eyes examined.Subjects were excluded from the study if they had previous oculardisease, previous ocular surgery, contact lens use, or systemic disease(e.g., diabetes). Three representative images were chosen for eachcorneal area and quantified using NeuronJ, a plugin for ImageJ software(NIH). Subbasal nerve count, nerve length, and nerve density, and mainnerve trunks, nerve branches, and total nerve fibers were determined foreach image. Statistical analysis was performed with ANOVA withBonferroni correction to compare the differences between the 5 corneaareas. A Pearson R coefficient and multivariate linear regressionanalysis were used to address the correlation with age and gender. Thedifferences were considered statistically different for p-value lessthan 0.05. Table 9 below shows the demographics for the subjects thatparticipated in this study.

TABLE 9 Demographics of healthy (control) subjects in study Total MEEIUI No. of patients (n) 85 37 48 Age (mean ± SD, yrs) 31.4 ± 10.5 30.0 ±11.5 32.6 ± 9.8 Gender (male/female) 35/50 17/20 18/30 Mean ± SEMTable 10 below lists the mean±SEM total nerve number, main nerve trunknumber, nerve branch numbers, total nerve length, main trunk nervelength, and branch nerve length for the central cornea and the fourperipheral quadrants of the cornea (inferior quadrant, inferiorquadrant, nasal quadrant, superior quadrant, and temporal quadrant).

FIG. 82 is a graph showing the mean main nerve trunk number(number/frame) in the central cornea and in each of the four peripheralcornea quadrants (inferior, nasal, superior, and temporal quadrants) inhealthy (control) subjects. FIG. 83 is a graph showing the mean mainnerve trunk length (μm²) in the central cornea and in each of the fourperipheral cornea quadrants (inferior, nasal, superior, and temporalquadrants) in healthy (control) subjects. FIG. 84 is a graph showing themean nerve branch number (number/frame) in the central cornea and ineach of the four peripheral cornea quadrants (inferior, nasal, superior,and temporal quadrants) in healthy (control) subjects. FIG. 85 is agraph showing the mean nerve branch length (μm/mm2) in the centralcornea and in each of the four peripheral cornea quadrants (inferior,nasal, superior, and temporal quadrants) in healthy (control) subjects.FIG. 86 is a graph showing the mean total nerve number (number/frame) inthe central cornea and in each of the four peripheral cornea quadrants(inferior, nasal, superior, and temporal quadrants) in healthy (control)subjects. FIG. 87 is a graph showing the mean total nerve length(μm/mm2) in the central cornea and in each of the four peripheral corneaquadrants (inferior, nasal, superior, and temporal quadrants) in healthy(control) subjects. The nerve parameters revealed an inverse correlationwith age (R=−0.20, p=0.017), but no correlation was found for gender andthe nerve parameters (p=0.781).

TABLE 10 Nerve measurements in healthy (control) subjects Nerve fibersCenter Inferior Nasal Superior Temporal Total nerve number 14.4 ± 0.58.9 ± 0.5  7.6 ± 0.4 9.1 ± 0.4 9.4 ± 0.5 (n/frame) Main trunk number 3.4 ± 0.1 2.1 ± 0.2  1.7 ± 0.1 2.3 ± 0.1 2.0 ± 0.1 (n/frame) Branchnumbers 10.5 ± 0.5 6.0 ± 0.4  5.3 ± 0.4 6.1 ± 0.4 5.9 ± 0.4 (n/frame)Total nerve length 18784.0 ± 434.1  11375.0 ± 1233.8  9980.1 ± 544.411973.0 ± 600.8  12115.3 ± 575.6  (μm/mm²) Main trunk nerve length8853.9 ± 255.8 5165.2 ± 395.3  4437.2 ± 317.6 5627.7 ± 377.8  5258.0 ±378.9  (μm/mm²) Branch nerve length 9456.0 ± 407.2 5470.3 ± 370.4 5043.2 ± 363.3 5641.0 ± 361.1  5567.3 ± 379.2  (μm/mm²) Mean ± SEM

The present study determined a central cornea nerve density in healthy(control) subjects of 18,784±4002 (μm/mm²) (mean±SD). Other previouslycalculated central cornea nerve densities (μm/mm²; mean±SD) in healthy(control) subjects include: 25,929; 6968; 21,600±5980; 20,300±6500; and19961.3±6552.9. The present study determined a mean subbasal nervedensity in each of the four peripheral cornea quadrants (μm/mm²;mean±SD) in healthy (control) subjects of: 11,375±5,994, inferiorquadrant; 9,980±5,019, nasal quadrant; 11,973±5,539, superior; and12,115±5,306, temporal quadrant. Other central and peripheral quadrantnerve densities (μm/mm²; mean±SD) in control (healthy) subjectsdetermined using Confoscan 2 (a slit-scanning confocal microscope) were:14,731±6,056, central; 8,477±6,460, inferior; 7,850±4,947, nasal;8,566±6,441, superior; and 12,556±6,909, temporal. These values andranges determined in healthy subjects (any of the values or rangesdescribed in this example) can be non-limiting references values orranges in any of the methods described herein.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1.-21. (canceled)
 22. A method of treating a subject having dry eyesyndrome, the method comprising: using in vitro confocal microscopy todetermine a number or average density of dendritic inflammatory cellspresent in the center of the cornea in an eye of the subject; comparingthe number or average density of dendritic inflammatory cells present inthe center of the cornea in the eye of the subject to a correspondingreference value; and selectively administering a topical steroidsolution to an eye(s) of a subject having dry eye syndrome, determinedto have an elevated number or density of dendritic immune cells presentin the center of the cornea as compared to the reference level; orselectively orally or topically administering two or moreimmunosuppressive agents to a subject having dry eye syndrome,determined to have no substantial change or a decreased number ordensity of dendritic immune cells present in the center of the cornea ascompared to the reference level.
 23. The method of claim 22, wherein thetopical steroid solution comprises a steroid selected from the groupconsisting of: loteprednol etabonate, dexamethasone, hydrocortisone,prednisolone, prednisone, methylprednisolone, betamethasone,dexamethasone, triamcinolone, beclometasone, fludrocortisone,deoxycorticosterone, and aldosterone.
 24. The method of claim 22,wherein the topical steroid solution contains loteprednol etabonate. 25.The method of claim 22, wherein the topical steroid solution isadministered at least once a day.
 26. The method of claim 22, wherein atleast one of the at least two immunosuppressive agents is a steroid. 27.The method of claim 26, wherein the steroid is selected from the groupconsisting of: loteprednol etabonate, dexamethasone, hydrocortisone,prednisolone, prednisone, methylprednisolone, betamethasone,dexamethasone, triamcinolone, beclometasone, fludrocortisone,deoxycorticosterone, and aldosterone.
 28. The method of claim 22,wherein at least one of the two immunosuppressive agents is selectedfrom the group consisting of: pimecrolimus, tacrolimus, sirolimus, andcyclosporine.
 29. The method of claim 22, wherein at least one of the atleast two immunosuppressive agents is selected from the group consistingof: pimecrolimus, tacrolimus, sirolimus, and cyclosporine.
 30. Themethod of claim 29, wherein the at least one immunosuppressive agent iscyclosporine.
 31. The method of claim 22, wherein the at least twoimmunosuppressive agents are administered at least twice a week.
 32. Themethod of claim 31, wherein the at least two immunosuppressive agentsare administered at least once a day. 33.-34. (canceled)
 35. The methodof claim 22, further comprising selecting a subject determined to havean elevated number or density of dendritic inflammatory cells present inthe center of the cornea in the eye of the subject as compared to thecorresponding reference value.
 36. A method of treating a subject, themethod comprising: using in vitro confocal microscopy to determine anumber or average density of dendritic inflammatory cells present in theperipheral cornea in an eye of the subject; comparing the number oraverage density of dendritic inflammatory cells present in theperipheral cornea in the eye of the subject to a corresponding referencevalue; and selectively topically or orally administering one or both ofat least one anti-inflammatory steroid or at least one immunosuppressiveagent to a subject determined to have an elevated number or elevatedaverage density of dendritic inflammatory cells present in theperipheral cornea as compared to the corresponding reference value. 37.The method of claim 36, wherein the subject is diagnosed as having eyeinflammation.
 38. The method of claim 36, wherein the at least oneanti-inflammatory steroid is selected from the group consisting of:hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone,dexamethasone, betamethasone, triamcinolone, beclometasone,fludrocortisone, deoxycorticosterone, and aldosterone.
 39. The method ofclaim 36, wherein the at least one immunosuppressive agent is acalcineurin inhibitor.
 40. The method of claim 39, wherein the at leastone calcineurin inhibitor is selected from the group consisting of:pimecrolimus, tacrolimus, sirolimus, and cyclosporine. 41.-42.(canceled)
 43. The method of claim 36, further comprising selecting asubject determined to have an elevated number or density of dendriticinflammatory cells present in the peripheral cornea in the eye of thesubject as compared to the corresponding reference value. 44.-46.(canceled)